Axon guidance proteins in neurological disorders Eljo Y Van Battum*, Sara Brignani*, R Jeroen Pasterkamp

Many neurological disorders are characterised by structural changes in neuronal connections, ranging from presymptomatic synaptic changes to the loss or rewiring of entire axon bundles. The molecular mechanisms that underlie this perturbed connectivity are poorly understood, but recent studies suggest a role for axon guidance proteins. Axon guidance proteins guide growing axons during development and control structural plasticity of synaptic connections in adults. Changes in expression or function of these proteins might induce pathological changes in neural circuits that predispose to, or cause, neurological diseases. For some neurological disorders, such as midline crossing disorders, investigators have identified causative mutations in genes for axon guidance. However, for most other disorders, evidence is correlative and further studies are needed to confirm the pathological role of defects in proteins for axon guidance. Importantly, further insight into how dysregulation of axon guidance proteins causes disease will help the development of therapeutic strategies for neurological disorders.

Introduction Pathological changes in neural circuitry are a unifying hallmark of many neurological diseases. In some diseases, structural connectivity defects are a result of the disease process, whereas in other disorders they have an early and causal role. The molecular mechanisms underlying perturbed structural connectivity are often poorly understood. Accumulating evidence from both human genetic and animal studies implicates axon guidance proteins in the rearrangement and loss of neuronal connections during neurological disease. Axon guidance proteins were originally identified as instructive cues to guide embryonic axons. These cues act as attractants or repellents for growing axons and guide them towards or away from specific regions in the developing brain or embryo.1,2 The concerted action of several different axon guidance proteins enables growing axons to find their targets. However, axon guidance proteins have many roles beyond embryonic axon guidance. These include functions in developmental processes, such as neuron cell body migration, but also roles in the adult nervous system—eg, in the control of synaptic plasticity. Human and animal studies have associated axon guidance proteins with neuronal connectivity changes in three classes of neurological disorders. First, mutations in genes encoding axon guidance proteins and receptors can cause congenital axon guidance disorders characterised by aberrant pathfinding of embryonic axons and neurons.3,4 Second, changes in axon guidance (receptor) genes and changed expression or function of these cues could contribute to the aetiology of multifactorial disorders such as autism, in which neuronal morphology and structural connectivity are affected during development.5–7 Third, subtle defects in axon guidance proteins could contribute to the development of late-onset brain disorders such as neurodegenerative diseases. These defects might increase disease susceptibility or might act as causative factors.8,9 In this Review, we highlight recent advances in understanding of how axon guidance proteins contribute to neurological diseases. We focus on the role of canonical axon guidance proteins and discuss examples in which defects in these

proteins mediate pathological changes in neuronal morphology and connectivity. Data from human studies are discussed, together with experiments in cellular and animal models, to explain disease mechanisms and to highlight opportunities for the development of therapeutic strategies. Defects in axon guidance proteins can also affect the cardiovascular and immune systems and thereby indirectly contribute to neurological disease. For example, in multiple sclerosis, axon guidance proteins mediate an aberrant immune response towards neuronal tissue. These non-neuronal effects are beyond the scope of our Review and are discussed elsewhere.10,11

Lancet Neurol 2015 Published Online March 11, 2015 S1474-4422(14)70257-1 *Authors contributed equally Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, Netherlands (E Y Van Battum MSc, S Brignani MSc, R J Pasterkamp PhD) Correspondence to: Dr R Jeroen Pasterkamp, Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht 3584 CG, Netherlands [email protected]

Axon guidance proteins Work in the past two decades has led to the discovery of a class of proteins that help to establish functional neural circuits through guidance of axons to their specific targets during development. These so-called axon guidance proteins are expressed along the trajectories of growing axons and are detected by a sensory structure at the axon tip—the growth cone (figure 1). Specialised receptor proteins at the growthcone-cell surface detect axon guidance proteins and trigger intracellular signalling cascades that cause axon steering through induction of changes in the growth-cone cytoskeleton. Axon guidance proteins can act as attractants or repellents—ie, either directing axons towards a specific structure or diverting them away from specific regions. Furthermore, they exist as both membrane-associated cues acting at short ranges or as secreted agents with longer range effects. Researchers have identified five families of canonical guidance proteins: Semaphorins, Netrins, Slits, Repulsive guidance molecules, and Ephrins (figure 1C).1,2 Semaphorins are a large family of proteins that includes both secreted (Semaphorin class three) and membraneassociated attractants and repellents (Semaphorins classes four to seven). In mammals, Semaphorins have been subdivided into five classes on the basis of sequence and structural similarities. The major growth-cone receptors for Semaphorins are members of the Plexin and Neuropilin protein families. Semaphorins and their receptors regulate many different aspects of neural circuit development, in both the central and peripheral nervous systems, including Published online March 11, 2015




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topographically organised axon connections between the eye and the brain.16 In addition to these canonical axon guidance proteins, additional protein families previously recognised for other functions have been implicated in axon guidance (eg, cell adhension molecules and morphogens).17–19 Many axon guidance proteins are pleiotropic and have diverse functions unrelated to axon guidance. This includes roles in other aspects of neural circuit development (eg, in axon fasciculation, pruning, and synaptogenesis), but also effects on non-neuronal cells in other organ systems (eg, in angiogenesis and bone formation).13,15,16,20

- - - - - - - - -- -- - -- --


Congenital disorders of aberrant pathfinding Netrin







6 7










Eph Intracellular

Figure 1: Axonal guidance and canonical axon guidance proteins (A) During embryonic development, axons are guided along predetermined routes by proteins in the extracellular environment. The growth cone, a sensory structure at the axon tip, enables axons to react to axon guidance proteins. Receptor proteins at the growth cone cell surface bind to the guidance cues and induce axon steering. Axon guidance cues can have repulsive (blue, –) or attractive (pink, +) effects on growing axons and exist as secreted proteins that form gradients in the extracellular matrix or as membrane-bound proteins with short-range effects. (B) Similar to axons, migrating cells in the developing nervous system are guided to their targets by repulsive and attractive axon guidance proteins. (C) Five families of canonical axon guidance proteins have been identified (top) that bind and signal through specific growth cone receptors (bottom). RGM=repulsive guidance molecules.

axon growth and guidance, axon bundling, and pruning.12 Two closely related secreted Netrin proteins have been identified in mammals, which signal through DCC receptors and UNC-5 receptors to induce attractive and repulsive effects, respectively. One of the best characterised effects of Netrins is their ability to attract axons to the midline of the nervous system during the formation of brain commissures.13 The Slit proteins (Slit1, Slit2, and Slit3) are secreted repellents that use proteins from the roundabout (Robo) family as receptors. Similar to Netrins, Slits have an important role during midline guidance. Once an axon has crossed the midline, repulsion by Slits prevents recrossing.14 Repulsive guidance molecules (RGMA-C) are glycosylphosphatidylinositol (GPI)-anchored repellents that signal via the Neogenin receptor. Although Repulsive guidance molecules remain poorly characterised, evidence is emerging that these cues mediate axon guidance events in different brain regions.15 EphrinA and EphrinB proteins act as short-range attractants or repellents. The EphrinA proteins (A1–A5) are tethered to the membrane via GPI anchors and interact with class A (A1–A8) Eph receptors; the EphrinB proteins (B1–B3) are transmembrane proteins that interact with class B (B1–B6) Eph receptors. Ephrins and Eph receptors are known for the development of 2

Principles of commissural axon guidance Commissural axon projections connect the left and right sides of the brain and the spinal cord to integrate information for coordinated sensory, motor, and cognitive functions. There are six major brain commissures (the anterior commissure, posterior commissure, corpus callosum, hippocampal commissure, habenular commissure, and the decussation of the superior cerebellar peduncle) and one spinal cord commissure (the anterior white commissure). In addition, several other axon bundles cross the midline, including the optic nerve and auditory pathway. Commissural axon guidance has been best studied for embryonic spinal commissural axons crossing the floorplate and for axons from pyramidal neurons in the cerebral cortex that form the corpus callosum (figure 2).21–23 Semaphorins and their Neuropilin receptors, Ephrins and Eph receptors, Netrins and DCC, and Slits and Robo receptors control midline guidance in different model organisms. Mutations in DCC or ROBO lead to midline guidance defects in humans. For a review of human genetic disorders of axon guidance caused by mutations in non-canonical guidance proteins we direct the reader to other articles.23,24

Congenital mirror movements Congenital mirror movements are pathological involuntary movements that mirror intentional movements on the contralateral side. Congenital mirror movements occur mostly in the forearms, hands, and fingers, and they can be a symptom of other brain disorders, such as Joubert’s syndrome. Non-syndromic congenital mirror movements is a very rare disorder that runs in an autosomal-dominant inheritance pattern in a few families worldwide, but can also present as a sporadic disease. Although congenital mirror movements can occur in healthy young children, they typically disappear by age 7 years. Imaging studies in patients have identified abnormal development of the corpus callosum and incomplete development, including abnormal decussation, of the corticospinal tracts, as anatomical defects underlying congenital mirror movements.3,4 Mutations in the Netrin-1 receptor DCC were recently reported to underlie non-syndromic congenital mirror Published online March 11, 2015



Crossing of embryonic spinal commissural axons


DCC Robo1*, Robo2, Robo3.1

Netrin Slit1–3

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DCC* Robo1, Robo2, Robo3.2


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Crossing of callosal axons Netrin


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CA IG DCC Nrp1, Robo1, Robo2


DCC, Robo1, Robo2


Draxin Slit1–3

Draxin Slit1–3

Draxin Slit1–3


Draxin Slit1–3 Post-crossing

Figure 2: Midline crossing of embryonic spinal commissural and callosal axons (A) The development of axon projections from spinal commissural neurons in the embryonic mouse dorsal spinal cord requires many different axon guidance proteins. Here, two classes of axon guidance proteins are shown: Netrin-1 and Slits. Netrin-1 attracts mouse spinal commissural axons expressing DCC towards the floorplate. As these axons approach the floorplate, Robo3.1 blocks Robo1 on the growth cone, and thereby blocks axon repulsion mediated by Slit proteins expressed at the floorplate, allowing axons to cross the midline. After midline crossing, Robo3.2 is expressed instead of Robo3.1. As a result, Robo1, Robo2, and Robo3.2 detect the Slit proteins, which repel the spinal commissural axons away from the floorplate. Netrin-1/DCC attraction is silenced by Robo1 to prevent recrossing of the spinal commissural axons. (Robo1* indicates inactivation by Robo3.1; DCC* indicates DCC silencing). (B) Callosal axons in the embryonic mouse brain originate from layer V pyramidal neurons in the cerebral cortex and project through the corpus callosum to connect both hemispheres. At the midline, callosal axons interact with axon guidance proteins that are expressed on the medial zipper glia and guidepost cells that bridge the two hemispheres. Neuronal guidepost cells located in the developing corpus callosum express Sema3C, which attracts callosal axons that express the Neuropilin-1 (Nrp1) receptor to the midline. Netrin-1–DCC signalling also attracts callosal axons to the midline. Axons are repelled and funnelled through the developing corpus callosum by glial guidepost cells in the indusium griseum and glial wedge that express Slits 1–3 and Draxin, and their receptors (Robo 1 and 2, and DCC, respectively). SCA=spinal commissural axons. FP=ventral floor plate. CA=callosal axons. IG=indusium griseum. CC=corpus callosum. GW=glial wedge. MZG=medial zipper glia.

movements in three unrelated families (table). Many of these mutations are predicted to produce truncated DCC proteins that do not have binding sites for Netrin-1 or do not have the intracellular domains needed for signalling.25,26 However, whether these truncated proteins are produced at all and whether they reach the plasma membrane still needs to be established. The ability of Netrin-1 and DCC to attract axons to the nervous system midline, together with the abnormal development of commissures in mice without DCC,72 support the hypothesis that in patients with congenital mirror movements who carry DCC mutations commissural axons do not reach and cross the midline because of reduced midline attraction. The finding that several other families and sporadic patients with congenital mirror movements do not have DCC mutations indicates that this is a genetically heterogeneous disorder.25,26,73 Axon guidance proteins and receptors with roles in midline guidance, other than DCC and ROBO3, such as Netrin-1, Slits, and Semaphorins, are potential candidate proteins that should be investigated. Further work is also needed to identify additional mutations in patients with

congenital mirror movements and to identify the molecular mechanisms through which DCC mutations cause these disorders.

Horizontal gaze palsy with progressive scoliosis Horizontal gaze palsy with progressive scoliosis is a rare disorder that presents with congenital absence of horizontal eye movement and scoliosis that develops during the first decade of life. It is an autosomal-recessive disorder that runs in around ten families worldwide and is caused by nonsense, frame-shift, splice-site, or missense mutations in ROBO3 (table).27 Electrophysiological and imaging studies show the absence of decussating axons in the pons and medulla. Uncrossed oculomotor and abducens intranuclear tracts are thought to account for the horizontal gaze.4 The cause of the scoliosis is not known but could be the result of asynchronous breathing.3,4 The heterogeneous nature of the mutations and their presence throughout the ROBO3 gene indicates a loss of ROBO3 function in patients. Two ROBO3 splice variants exist: ROBO3.1 and ROBO3.2. The ROBO3 mutations reported in horizontal Published online March 11, 2015



Study type

Disease mechanism linked to defect in axon guidance gene*

GWAS; affected-only linkage analysis in 11 patients of two unrelated families;25 linkage analysis in 13 patients from three unrelated families26

Abnormal development and crossing of corpus callosum and corticospinal tracts

Genetic disorders of midline crossing Congenital mirror movements DCC

Horizontal gaze palsy with progressive scoliosis ROBO3

Linkage analysis in 25 patients from eight unrelated families;27 DNA sequence Abnormal decussation of axons in the pons and medulla analysis of two patients from two unrelated familes;28 DNA sequence analysis of seven patients from five unrelated families;29 linkage analysis in ten patients from four unrelated families30

Dyslexia ROBO1

Case report;31 polymorphism screening in 19 patients from one family;31 functional magneto-encephalography study;32 association study in cohort of 1177 individuals of 538 families;33 association study in 804 patients of 493 families (1727 total sample);34 and association study in 1416 individuals from 421 families and association study in 749 individuals from 101 families35

A specific haplotype31,32 decreases Robo1 protein expression, which leads to crossing defects in the auditory pathway


Association study in 180 twins and their parents36

Possibly defects in visual pathways


Comparative genomic hybridisation array in 386 unrelated patients;37 case report, 1 patient selected from 48 patients with no mutations in known responsible genes;38 Sanger sequencing, SNP analysis in 66 unrelated patients39

Failure of GnRH neurons to migrate to the hypothalamus during development


Sanger sequencing, SNP analysis in 66 unrelated patients39


Genetic disorders of neuron migration Kallmann’s syndrome

Hirschsprung’s disease SEMA3A

SNP analysis in 119 patients and 93 controls;40 mutational screening in 200 patients Defects in enteric neural precursor migration and 200 controls41


Mutational screening in 200 patients and 200 controls41



GWAS in 169 Chinese Han family trios42



GWAS, a trio association study using DNA from 252 families recruited to AGRE43



Family-based autism GWAS of 597 families and a second existing autism GWAS of 696 families from AGRE44



Expression study, samples from anterior cingulate cortex of eight patients and 13 controls45



Expression study (mRNA from Epstein–Barr virus transformed B lymphocytes from six unrelated patients and six controls)46



Genome-wide copy number variation in 286 patients coming from four distinct populations of patients with autism47



Expression study (entorhinal cortex) in rats with electrically induced epileptogenesis48

Mossy fibre sprouting into molecular layer of the dentate gyrus


Functional study (kindling rat model)49

Application of Ephrin A and EphA agonists and antagonists modulates mossy fibre sprouting and seizure development


Expression study (hippocampus) in mouse model of temporal lobe epilepsy by intraperitoneal administration of pilocarpine50



Expression study (hippocampus) in 30 rats with kainic-acid-induced status epilepticus51



Expression study in temporal neocortex of 35 epileptic patients and 15 controls, and 54 rats (control or in which epilepsy was induced by lithium-pilocarpine administration)52



Genome-wide copy number variation in 117 Turkish patients with ALS and 109 matched healthy controls53



Expression study in human blood (120 patients and 58 controls);54 DNA sequencing of a patient with sporadic ALS;54 functional study in animal models: mutant SOD1 zebrafish, hSOD-G93A rats and hSOD1-G93A mice54

EPHA4 mutation is significantly protective for ALS, and leads to a long survival; genetic as well as pharmacological inhibition of Epha4 signalling rescues the mutant SOD1 phenotype in zebrafish and increases survival in models of ALS

Autism spectrum disorders and epilepsy Autism spectrum disorders


Neurodegenerative diseases ALS

(Table continues on next page)

4 Published online March 11, 2015


Study type

Disease mechanism linked to defect in axon guidance gene*


Expression study (neuromuscular junction of hSOD-G93A mouse model55)

Axon retraction or inhibition of axonal sprouting at neuromuscular junction


GWAS on 250 patients with sporadic ALS and 250 controls from Chinese Han population56



GWAS on 2 groups (443 sibling pairs discordant for Parkinson’s disease; 332 matched case-unrelated control pairs) of USA or European origin57



GWAS on two groups (443 sibling pairs discordant for Parkinson’s disease; 332 matched case-unrelated control pairs) of USA or European origin;58 metaanalysis of 12 studies in different populations (3539 cases and 3250 controls)59



GWAS on two groups (443 sibling pairs discordant for Parkinson’s disease; 332 matched case-unrelated control pairs) of USA or European origin60



Highest ranked SNPs in genomic pathway analyses (meta-analysis of multiple studies)60,61



Expression study (substantia nigra of four patients with Parkinson’s disease vs controls)62



Expression study in post-mortem hippocampal tissue from three patients and three controls;63 expression study in hippocampus from Alzheimer’s disease mouse model overexpressing human APP with familial swe/ind mutations;63 functional study in APPswe/PSEN1ΔE9 transgenic mice64

Binding of Aβ to EPHA4 induces synaptic dysfunction or synaptic loss


Functional study in APP mutant mice in a mixed background of C57BL6, 129SvEv, and 129 Ola65

Netrin-1 binds APP and regulates cleavage of Aβ from APP


Functional study in hAPP line J20 mouse model66

Increasing EphB2 levels or function could be beneficial in Alzheimer’s disease


Multiprotein complex from the hippocampus of patients with Alzheimer’s disease67,68



Expression study (posterior cingulate area of seven patients with sporadic early onset Alzheimer’s disease, seven patients with familial monogenic PSEN1 Alzheimer’s disease, and seven controls)69



GWAS in 6992 patients and 13 472 controls (meta-analysis)70,71


(Continued from previous page)

Parkinson’s disease

Alzheimer’s disease

For more information see appendix. GnRH=gonadotropin-releasing hormone. GWAS=genome-wide association study. Aβ=amyloid β. APP=amyloid precursor protein. AGRE=Autism Genetic Resource Exchange. ALS=amyotrophic lateral sclerosis. swe/ind=Swedish/Indiana. *Disease mechanisms proposed on basis of combined data from human genetics, imaging, expression, and animal studies.

Table: Canonical axon guidance proteins in neurological disease

gaze palsy with progressive scoliosis affect both splice variants, and midline crossing defects are probably caused by the inability of axons to block their axonal Slit responses, preventing axons from crossing the midline. Recently, conditional knockout mice have been generated to selectively ablate Robo3 expression in mouse hindbrain neurons.74 These mice will be instrumental to establish the causal mechanisms of horizontal gaze palsy with progressive scoliosis.

Dyslexia Dyslexia has a prevalence of about 5% of school-age children. Twin and family studies show a strong genetic basis for dyslexia, and several genomic loci harbour candidate dyslexia susceptibility genes. Several of these genes encode for axon guidance proteins or receptors, including SEMA4F and ROBO1 (table).75 In a Finnish family, a rare, weakly expressing haplotype of ROBO1 was reported to cosegregate with dyslexia in a dominant manner.31 Dyslexia associates with various auditory disorders, and electrophysiological assessment of the Finnish

family revealed crossing defects in the auditory pathway in family members with dyslexia.32 This, together with the observation that Robo1 knockout mice show various midline crossing defects,76 suggests that normal crossing of auditory pathways in human beings might need ROBO1 expression. However, no differences were shown in ROBO1 expression between healthy individuals versus dyslexic individuals and other studies could not replicate the association between ROBO1 and dyslexia in human beings (table).31,77 Therefore, further studies are needed to assess whether or not dysregulation of ROBO1 contributes to the pathogenesis of dyslexia.

See Online for appendix

Genetic disorders of neuron migration Axon guidance proteins in cell migration Axon guidance proteins not only guide growing axons but also guide migrating cells (figure 1B). Newly differentiated neurons and glial cells use axon guidance proteins as directional cues in the developing nervous system.78 Members of all five families of canonical axon guidance proteins have been implicated in neural Published online March 11, 2015



migration, and changes in their expression or function are linked to disorders characterised by aberrant cell migration.

Kallmann’s syndrome Kallmann’s syndrome affects about one in 8000 men and one in 40 000 women, and is characterised by congenital anosmia and hypogonadotropic hypogonadism. In patients with Kallmann’s syndrome, neurons that produce gonadotropin-releasing hormone (GnRH) do not reach the A GnRH neuron migration

AO AOB OB GnRH neuron OB OE OB SEMA3A mutation Sema7A Sema777A


Sema3A VNO

hypothalamus, resulting in an absence of sexual development during puberty. Mutations in several different genes have been linked to Kallmann’s syndrome; many of these genes are implicated in axon growth and guidance.79 During embryonic development, GnRH neurons use olfactory axons as a scaffold to reach the hypothalamus (figure 3A). It is proposed that in a subset of patients with Kallmann’s syndrome, defects in olfactory axon guidance cause misrouting of GnRH neurons. Several loss-offunction mutations in SEMA3A and some rare mutations in SEMA7A have been noted in studies of patients with Kallmann’s syndrome (table).37,38 Knockout mice provide valuable insight into the possible disease process in patients with Kallmann’s syndrome who carry these mutations. Knockout mice for Sema3A or functional Sema3A receptors have a Kallmann’s syndrome-like phenotype, with olfactory axon guidance defects, aberrant GnRH neuron migration, and reduced fertility.37,80 In these mice, olfactory axon misrouting directs GnRH neurons to regions of the brain other than the hypothalamus (figure 3A).37,80,81 By contrast, Sema7A is not needed for the guidance of olfactory axons but does act as a molecular signal on olfactory axons to aid GnRH neuron migration. In the absence of Sema7A, GnRH neurons detach from olfactory axons and fail to migrate into the brain (figure 3A).82 Although further work is needed to establish how mutations in SEMA3A and SEMA7A linked to

SEMA7A mutation


Enteric nervous system development

RET mutation

Healthy embryo ↓RET


RET+ S-NCC Sema3A SEMA3A mutation




Figure 3: Axon guidance proteins and human genetic disorders of neuron migration (A) Head of a mouse embryo. The secreted semaphorin Sema3A (blue gradient) is expressed along the trajectory of olfactory axons from the olfactory epithelium, which project to the OB and the VNO, which project to the AOB. The membranebound semaphorin Sema7A is expressed on the olfactory axons. GnRH neurons (in green) in the developing olfactory system migrate along olfactory axons to the OB and then along central VNO to the hypothalamus. Mutations in SEMA3A are proposed to lead to defects in olfactory axon guidance. As a result, GnRH neurons stall in the nasal compartment or follow misrouted olfactory axons into abnormal regions of the brain. Sema7A is required on olfactory axons to promote the migration of GnRH neurons. SEMA7A mutations can cause GnRH neurons to detach from olfactory axons and accumulate in the nasal compartment. (B) During the development of the mouse enteric nervous system, enteric neural precursor cells (green) are derived from either the vagal neural crest or the sacral neural crest. Enteric neural precursor cells derived from vagal neural crest cells exit the hindbrain area and migrate to the developing intestinal tract. Once at the intestinal tract, these cells express the RET receptor, which promotes proliferation, migration, and differentiation. The gut mesoderm expresses GDNF, which is a substrate of RET coreceptor GFRα1. RET signalling drives the innervation of the intestinal tract by vagal neural crest-derived cells in a rostral to caudal direction. Sema3A is expressed in the distal gut (blue). Initially, repulsion of enteric neural precursor cells derived from S-NCC by Sema3A prevents entry of these cells into the intestinal tract. After a delay until later development, Sema3A expression is reduced, allowing the innervation of the gut by the sacral neural crest-derived cells in a caudal to rostral direction. Mutations in RET cause Hirschsprung’s disease. RET deficiency results in decreased innervation of the gut by both populations of enteric neural precursor cells, leading to partial enteric agangliosis. A subset of patients with Hirschsprung’s disease have SEMA3A mutations and display enhanced expression of SEMA3A (blue) in the gut. This prevents or delays the entry of sacral neural crest-derived enteric neural precursor cells into the intestinal tract. OE=olfactory epithelium. OB=olfactory bulb. VNO=vomeronasal axons. AOB=accessory olfactory bulb. GnRH=Gonadotropin-releasing hormone. GDNF=glial derived neurotrophic factor. V-NCC= vagal neural crest cells. S-NCC=sacral neural crest cells. Published online March 11, 2015


Kallmann’s syndrome affect the function of these proteins, these results support an important role for Semaphorins in the pathogenesis of Kallmann’s syndrome. So far, disease-causing mutations have been identified in roughly 30% of patients with Kallmann’s syndrome, but more genetic studies are needed. Several other axon guidance proteins have been implicated in GnRH neuron migration—eg, Slit2 and Sema4D—and are candidate proteins for Kallmann’s syndrome.83,84

Hirschsprung’s disease Hirschsprung’s disease is characterised by a congenital absence of ganglia containing enteric neurons along variable lengths of the distal intestinal tract. This causes tonic contraction, intestinal obstruction, and bowel distention.85 Agangliosis is attributed to a defect in the time-dependent migration of neural-crest-cell-derived enteric neural precursors to the intestinal tract. Hirschsprung’s disease is rare and affects about 0·01% of the European population.86 Most cases of Hirschsprung’s disease are caused by heterogeneous mutations in RET, a proto-oncogene that causes total intestinal agangliosis when mutated in mice (figure 3B).87 SEMA3A and SEMA3D mutations have been identified in some families with Hirschsprung’s disease and a subset of patients show increased SEMA3A expression in affected colon tissue (table).41,88 Normally, SEMA3A is expressed in the mesenchyme of the distal intestine and acts as a repulsive signal for migrating enteric neural precursor cells from the sacral neural crest. In Sema3Aknockout mice, enteric neural precursor cells from the sacral neural crest enter the distal gut prematurely.89 Based on these findings, researchers have proposed that enhanced expression of SEMA3A in patients with Hirschsprung’s disease inhibits the migration of sacral neural crest enteric neural precursor cells into the developing distal intestine (figure 3B). Several patients with SEMA3A mutations have cosegregating RET mutations and it has been suggested that both mutations work synergistically.89 Both populations of enteric neural precursor cells might be affected in patients with the two mutations, compared with single mutation carriers, leading to a more severe disease phenotype.

Autism spectrum disorders Neurodevelopmental disorders such as autism spectrum disorders and attention-deficit hyperactivity disorder have an onset during early childhood and are caused by largely uncharacterised defects in neural development. Genetic evidence that implicates defective axon guidance protein function in neurodevelopmental disease is still sparse, but at the moment is strongest for autism spectrum disorders. Patients with autism spectrum disorders show impaired social and communicative skills and repetitive behaviours. Genetic risk contributes to the disorders, but the underlying genetic alterations remain unknown in

most cases.90 However, some human genetic, imaging, and neuropathological studies have identified connectivity as a converging neural substrate. It is generally believed that autism spectrum disorders are caused by a decrease in connectivity between specific brain regions (hypoconnectivity) together with an increase in connectivity within regions (hyperconnectivity). The hyperconnectivity phenotype includes an increase in the number of neurons, dendritic branches, and spines, whereas reduced connectivity between brain regions might arise from defective axon growth and guidance (figure 4A).5–7 These phenotypes could arise from defects in axon guidance proteins, and human genetic and tissue profiling studies have begun to identify axon guidance genes as susceptibility factors to autism spectrum disorders (table). In addition, expression of axon guidance proteins and receptors is decreased in post-mortem brain tissue or blood in patients with autism (table). However, functional evidence to link these findings to autism spectrum disorders is largely absent.

Epilepsy Genetic risk factors have an important role in epilepsy, but evidence to support a causal role for genetic defects in axon guidance genes is lacking. Rather, the strongest support for a role for axon guidance proteins in epilepsy is from animal studies. Seizure-induced changes in expression of axon guidance proteins have been shown in experimental models of epilepsy (table)50–52,91 and could cause the rewiring of neuronal circuits during later stages of epilepsy, contributing to its progressive and chronic nature. In the epileptic hippocampus, seizures lead to a progressive remodelling of the axons of dentate gyrus granule cells, known as mossy fibres. Normally mossy fibres extend towards the CA3 region and form synapses on the dendrites of CA3 pyramidal neurons (figure 4B). However, as a consequence of recurrent seizures, dentate gyrus granule cells can generate new mossy fibre collaterals, which aberrantly innervate the molecular layer and synapse with their own dendrites (figure 4B). This change leads to a recurrent excitatory circuit that causes the spontaneous recurrence of focal seizures and propagation of an epileptic state.92 Semaphorins have been linked to this rewiring event in rodent models of epilepsy.48,51 Sema3A is a repellent for mossy fibres and is normally delivered from the axons of neurons in the entorhinal cortex to the outer part of the molecular layer. In a rat model for temporal lobe epilepsy, Sema3A is downregulated in neurons in the entorhinal cortex, reducing the repulsive nature of the molecular layer. This reduction might allow ingrowth of mossy fibre collaterals and the formation of aberrant synapses in a recurrent excitatory circuit (figure 4B).48 Whether analogous expression changes occur in temporal lobe epilepsy in human beings and whether in-vivo manipulation of Sema3A function can alter mossy fibre sprouting await further study. The EphrinA5–EphA5 ligand–receptor Published online March 11, 2015



system might also contribute to functional mossy fibre rewiring, because infusion of EphrinA and EphA antagonists modulates the extent of mossy fibre sprouting and seizure activity, although the mechanism is unknown.49 Furthermore, the effect of regulation of axon guidance proteins in network remodelling in epilepsy might not be limited to mossy fibre sprouting; several other regions, such as CA1 and CA3, of the rodent and human hippocampus display seizure-induced changes in expression of axon guidance proteins (table). Epilepsy is not only characterised by marked changes in neuronal connectivity but also by the abnormal migration and location of hippocampal neurons.93 Given the role of axon

guidance proteins in cell migration, it is tempting to speculate that dysregulation of axon guidance proteins could contribute to epilepsy-associated migration defects.

Neurodegenerative diseases Neuronal connectivity and neurodegenerative diseases Accumulating evidence indicates that changes in neuronal connectivity are not only a result of the neurodegenerative disease process but could also have a causative role in disease susceptibility, onset, or progression. For example, in many neurodegenerative diseases morphological and functional changes in axons and synapses precede symptom onset.94,95 Dendritic spine


Afferent axon Dendrite Cell body

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Autism spectrum disorder

Healthy hippocampus EC axons

Nrp1 PlexinA4?

Molecular layer

Mossy fibres


Pyramidal cells

Epileptic hippocampus EC axons

Cue X Granule cells

Mossy fibre collaterals

DG Sema3A Mossy fibres CA3

Granule cells

Pyramidal cells



Figure 4: Axon guidance proteins in autism spectrum disorders and epilepsy (A) Autism spectrum disorders are characterised by a reduction in axonal connections between specific brain regions and an increase in connectivity within brain regions, including an increase in the number of neurons, dendritic branches, and spines. (B) Adult mouse hippocampus. DG granule cells (green) in the hippocampus normally project mossy fibre axons towards CA3, making synaptic contacts (yellow) with the dendrites of pyramidal cells (blue). Neurons in the entorhinal cortex innervate the outer part of the molecular layer and secrete Sema3A, which repels mossy fibres in the molecular layer through interaction with Neuropilin 1 (Nrp1), and possibly Plexin A4. In experimental models of temporal lobe epilepsy, Sema3A expression is downregulated in EC neurons and the molecular layer. This could allow newly formed mossy fibre collaterals (red), generated as a result of cell death or seizure activity, to invade the molecular layer and form aberrant synapses on DG granule cells. A reduction in the expression of unknown axon guidance proteins in CA3 is also proposed to contribute to the formation of aberrant mossy fibre synapses in this region of the hippocampus. EC=entorhinal cortex. DG=dentate gyrus.

8 Published online March 11, 2015


Genome-wide association, microarray profiling, and proteomics studies link axon guidance proteins to neurodegenerative diseases (table). Given their role in maintenance of axonal and synaptic connections in human adults,96,97 changes in axon guidance proteins could possibly trigger defects in neuronal networks, such as synaptic changes, which lead to neuronal dysfunction and loss in patients with neurodegenerative disease.


Terminal Schwann cell Axon terminal

Neuromuscular junction

Sema3A↑ Axon retraction or impaired axonal sprouting - -

Amyotrophic lateral sclerosis Although progress has been made to determine the genetic basis of amyotrophic lateral sclerosis, the mechanism of how genetic changes cause the disease is incompletely understood.98 In amyotrophic lateral sclerosis, the most distant cellular sites of the motor neuron (ie, distal axons and synapses) are affected first during early stages of the disease.94 It is therefore thought that subtle changes in neuronal connections might be causally related to the disease process, perhaps in conjunction with other causal or susceptibility factors. Several pieces of experimental evidence support the idea that dysregulation of axon guidance proteins might trigger the denervation of neuromuscular junctions, the synapses connecting motor neuron axons to muscle fibres (table). SOD1G93A mice, a commonly used model for amyotrophic lateral sclerosis, have a marked increase in the expression of Sema3A in terminal Schwann cells, which surround neuromuscular junctions (figure 5A). This increase is greatest for terminal Schwann cells surrounding motor nerve terminals (axons innervating fast-fatigable or type IIb/IIx muscle fibres), which have a low intrinsic ability for compensatory nerve sprouting after injury and that are lost first in SOD1G93A animal model.55 Researchers have therefore proposed that increased expression of Sema3A inhibits motor axon sprouting or triggers de-adhesion or repulsion of motor axons at the neuromuscular junctions, leading to muscle denervation and motor neuron degeneration. This model of denervation induced by axon guidance proteins is also supported by work on EPHA4.54 Gene knockdown and pharmacological inhibition of EphA4 rescue the axon outgrowth and branching defects observed in mutant SOD1 transgenic zebrafish. Similarly, genetic deletion of one EphA4 allele in SOD1G93A mice reduces muscle denervation and motor neuron loss, and significantly prolongs survival. Knockdown of EphA4 also rescues axonopathies induced by expression of TDP43, another protein that can cause amyotrophic lateral sclerosis, and by knockdown of SMN1 (survival motor neuron 1), the causative gene for spinal muscular atrophy. Thus, EPHA4 might be a generic therapeutic target in several motor neuron diseases. In patients with amyotrophic lateral sclerosis, EPHA4 expression is inversely correlated with age at disease onset and survival; loss-of-function mutations in EPHA4 are associated with longer survival.54 Motor neurons that are most vulnerable to degeneration (innervating fast-


Muscle fiber

Myelin Motor axon



- -


Terminal Schwann cell





Nogo-A↑ Muscle



Sema3A Netrin-1

Healthy individual



Alzheimer’s disease


Microtubule Tubulin Microtubule polymerisation

Aβ Netrin-1 Cdk5


P GSK-3β P

X Decreased microtubule polymerisation

Axon repulsion Synapse stabilisation


X GSK-3β Decreased microtubule polymerisation


Axon repulsion Retraction of synaptic contacts Neuron loss

Figure 5: Axon guidance proteins in neurodegenerative disease (A) Axons of motor neurons form specialised synapses, called neuromuscular junctions, on muscle fibres. These synapses are surrounded by terminal Schwann cells (left panel). In the right panel, G93A-hSOD1 mice, a mouse model for amyotrophic lateral sclerosis, have a marked increase in the expression of the motor axon repellent Sema3A in terminal Schwann cells. In addition, expression of the neurite growth inhibitor Nogo-A is increased in skeletal muscle and concentrations of EphA4, a receptor for the repulsive ephrinA protein family, are increased in experimental models of and in patients with amyotrophic lateral sclerosis. These molecular events are proposed to participate in the establishment of a repulsive environment at the neuromuscular junction, causing axon retraction or impaired axonal sprouting. (B) Collapsin response mediator protein (CRMP)2 is a cytosolic protein that binds Tubulin dimers, and promotes microtubule polymerisation and consequently axon elongation. Following binding of Sema3A to its Nrp1/PlexinA receptor, CRMP2 is recruited to the PlexinA receptor and becomes sequentially phosphorylated at Ser522 and Thr509 by Cdk5 and GSK-3β kinases, respectively. The phosphorylated form of CRMP2 shows a reduced association with Tubulin. Therefore, microtubule polymerisation is inhibited upon Sema3A exposure, inducing growth cone collapse and axon repulsion. In Alzheimer’s disease, CRMP2 is hyperphosphorylated. This might induce enhanced repulsive signaling by Sema3A and retraction of synaptic contacts and neuron loss. Aβ phosphorylates CRMP2 in a RhoA GTPase-dependent manner. Netrin-1 binds APP and might inhibit the cleavage of APP into Aβ. Reduced expression of Netrin-1 leads to increased Aβ levels and an exacerbation of Alzheimer’s-disease-like phenotypes in mouse models. Aβ=amyloid β. APP=Amyloid precursor protein. Published online March 11, 2015



fatigable or type IIb/IIx muscle fibers)and have low intrinsic ability for disease-induced neuromuscular reinnervation express higher levels of EphA4 in SOD1G93A transgenic mice. These observations suggest that high concentrations of axonal EphA4 might inhibit motor axon sprouting or cause deadhesion of neuromuscular junctions through interactions with repulsive EphA4 ligands, such as the EphrinA proteins, in the neuromuscular junctions or muscle (figure 5A). The neurite growth inhibitory molecule Nogo-A has been suggested to also have axon growth inhibitory effects at the neuromuscular junction. Patients with amyotrophic lateral sclerosis and SOD1 transgenic mice show an upregulation of Nogo-A in skeletal muscles.99 Although further work is needed to establish how changes in Nogo-A expression contribute to the pathogenesis of amyotrophic lateral sclerosis, investigators have shown that application of anti-Nogo-A antibodies delays disease progression in SOD1G93A transgenic mice.100 These observations have triggered the development of a humanised anti-NOGO-A antibody (ozanezumab), which is now being tested in a phase 2 clinical trial for application in patients with amyotrophic lateral sclerosis (NCT01753076). Collectively, these data support a model in which dysregulation of axon guidance proteins in amyotrophic lateral sclerosis contributes to motor axon retraction or impaired axonal sprouting, leading to muscle denervation and motor neuron death. Important goals for future research are to provide further functional data to support this model and to establish whether or not other axon guidance proteins associated with amyotrophic lateral sclerosis101–103 have similar roles in the disease process.

Parkinson’s disease Gene expression profiling in the substantia nigra pars compacta of patients with Parkinson’s disease or in experimental animal models of Parkinson’s disease shows differences in the expression of axon guidance proteins and receptors (table). In addition, genomic pathway analyses have identified axon guidance pathways in the aetiology of Parkinson’s disease (table). Although axon guidance proteins play a crucial part in the development of dopaminergic circuits,104 whether and how these expression and genetic changes are functionally linked to Parkinson’s disease needs to be established. Understanding of the potential role of these cues in Parkinson’s disease will need further analysis of their role in the dopaminergic system. This information could also help to improve cell replacement strategies in patients with Parkinson’s disease. The transplantation of healthy dopaminergic neurons into the brains of patients is a promising approach to alleviate the progressive symptoms of Parkinson’s disease.105 Transplanted dopaminergic neurons often establish insufficient and inappropriate axon connections leading to side-effects in patients. A better understanding of the 10

axon guidance mechanisms that normally wire the dopaminergic system could help molecular strategies to direct the axons of transplanted dopaminergic neurons to appropriate regions of the brain.

Alzheimer’s disease A recent whole-genome-expression profiling study in patients with familial and sporadic early-onset Alzheimer’s disease showed changes in the expression of several axon guidance proteins (table).69 Although this work implicates the axon guidance pathway in Alzheimer’s disease, early evidence of a pathological role for axon guidance proteins came from the isolation of a protein complex from the hippocampus of patients with Alzheimer’s disease that contained SEMA3A and collapsin response mediator protein (CRMP)2.67 CRMP2 is a microtubule-assembly factor downstream of Sema3A. Sequential phosphorylation of CRMP2 at ser522 and thr509 by the protein kinases Cdk5 and GSK-3β, respectively, reduces its interaction with tubulin and is required for axon repulsion induced by Sema3A (figure 5B). Neurofibrillary tangles contain a hyperphosphorylated form of CRMP2 that has increased phosphorylation of several aminoacids, including ser522 and thr509.106 This hyperphosphorylated state might possibly potentiate Sema3A signalling, leading to increased axon repulsion, retraction of synaptic contacts, and neuron loss. Amyloid β (Aβ) increases the phosphorylation of CRMP2 through a RhoA GTPasedependent mechanism.68 This system hints at cross-talk between Aβ and Sema3A signalling in Alzheimer’s disease. Axon guidance proteins can function both upstream and downstream of Aβ. Netrin-1 can bind the Aβ region of amyloid precursor protein and might negatively regulate cleavage of amyloid precursor protein.65 This is supported by the observation that models of Alzheimer’s disease with deletion of one Netrin-1 allele have significantly increased Aβ concentrations. Conversely, intracerebroventricular injection of Netrin-1 decreases Aβ concentrations in transgenic mice and improves memory function.65 Another interesting link between Netrin-1 and Alzheimer’s disease is the observation that processing of DCC by the protease Presenilin-1 is needed for Netrin-1-mediated axon guidance.107 Mutations in Presenilin-1 are a cause of familial Alzheimer’s disease. Therefore, in addition to contributing to defects in amyloid precursor protein processing, Presenilin-1 mutations could lead to changes in Netrin-1 signalling. In addition to Netrin-1, Aβ also interacts with EphB2 and EphA4. In mouse models of Alzheimer’s disease, Aβ directly binds EphB2, inducing the internalisation and degradation of the receptor. This causes a reduction in the cell surface expression of the NMDA glutamate receptor and leads to deficits in synaptic plasticity and cognitive function.66 Increases in EphB2 concentrations in the hippocampus of mice in models of Alzheimer’s Published online March 11, 2015


disease reverses their cognitive and behavioural deficits associated with Alzheimer’s disease.63,65 Similarly, Aβ binds to and signals via EphA4 to induce synaptic damage. The synaptotoxic effects of Aβ oligomers can be reduced by in-vivo knockdown of EphA4 expression.64 These data indicate that Netrin-1 and Eph receptors could have promise as potential therapeutic targets to modulate the concentration and effects of Aβ in Alzheimer’s disease.

Conclusions and future directions Because of their prominent role in neural circuit development, investigators have long considered changes in the expression and function of axon guidance proteins as potential causative events in neurological diseases with a developmental origin. Indeed, genetic studies have identified specific mutations in axon guidance receptors that cause congenital disorders of axon guidance such as congenital mirror movements and horizontal gaze palsy with progressive scoliosis. So far only few disorders have been reported, and in most cases their molecular and cellular causal mechanisms remain poorly understood. The small number of disorders directly linked to mutations in axon guidance genes could be explained by the proteins’ crucial role in cellular processes such as heart development, angiogenesis, and bone formation. Mutations in some axon guidance genes might simply not be compatible with life. For congenital mirror movements and horizontal gaze palsy with progressive scoliosis, findings in mouse models have clearly established a causal role for mutations in DCC and ROBO3 receptor genes, respectively. This evidence base contrasts with that for most other neurological disorders, for which evidence for the involvement of specific axon guidance proteins is correlative and based on gene expression studies or genome-wide association studies. In these cases functional studies that use molecular approaches and genetic mouse models for disease-specific mutations, are needed to establish causality. However, even in diseases such as congenital mirror movements and horizontal gaze palsy with progressive scoliosis, further molecular and cellular studies are needed to establish the precise effects of the reported DCC and ROBO3 mutations. Often mutations occur throughout the entire protein and probably impair different properties of the protein such as ligand binding or dimerisation. Understanding of the mechanism of action of specific mutations will help to design targeted therapeutic strategies. Advances in genetic, neuroimaging, and electrophysiological techniques are needed to identify and characterise neural circuit malformations caused by defects in axon guidance proteins in undiagnosed neurological patients. This analysis can be guided by work in animal models. Anatomical analyses of mouse models with disease-associated mutations in, or knockout models for, specific axon guidance proteins will not only further understanding of the role of the axon guidance proteins in neurological disease but also present a means for the in-depth identification of axon tract and

Search strategy and selection criteria We searched PubMed with the terms “axon guidance and neurological disease”, by combining the term “axon guidance” with the various diseases covered in this Review (eg, Alzheimer’s disease), and by combining a specific class of axon guidance proteins (eg, semaphorin) with a specific disease. We searched for reports published from Jan 1, 1990 to May 1, 2014. We also manually searched the reference lists of published work to identify further relevant references. Only papers published in English were considered. The final reference list was generated on the basis of originality and relevance to the broad scope of this Review. When many papers addressed the same topic, one or more were cited as representative of the issue.

connectivity defects that characterise the diseases. These features can subsequently be used to screen populations of patients and identify those with mutations in similar or related genes. The feasibility of this approach can be inferred from midline guidance disorders such as congenital muscle movements or horizontal gaze palsy with progressive scoliosis for which patients with mutations in DCC and ROBO have similar midline guidance defects to mice with the equivalent genetic defects. The role of axon guidance proteins in multifactorial neurological diseases remains incompletely understood. In some diseases, such as epilepsy, axon guidance proteins might be dysregulated as a result of the disease process and contribute to disease progression, severity, or duration. In other complex diseases, such as autism spectrum disorders, defects in axon guidance genes might have a causative role but the functional contribution of changes in individual proteins is often small. For example, in patients with Parkinson’s disease, consistent replication of the associations between individual single nucleotide polymorphisms in axon guidance genes and disease onset, duration, or severity has been difficult to achieve. By contrast, genomic pathway analyses that take into account several different single nucleotide polymorphisms indicate a clear role for the axon guidance pathway. Therefore, a combination of several small effects, caused by variants in different axon guidance genes, is probably needed to reach a level of biological dysfunction that triggers disease. Many of the observations that link axon guidance proteins to neurological disease (eg, changes in gene expression) are still correlative and should be interpreted with caution. They might simply be changes that are a result of the disease or just markers of the disease and but not contribute to its aetiology. Further cellular and animal studies are needed to test the functional contribution of these variants. Recent progress in stem cell biology that enables the conversion of somatic cells, such as skin fibroblasts, into stem cells (induced pluripotent stem cells [iPSC]) and, Published online March 11, 2015



subsequently, into differentiated neurons, is an invaluable method to further study the disease mechanisms by which defective axon guidance proteins cause neurological disease. For example, recent studies have used iPSC technology to transduce patient-derived fibroblasts into neurons to study synaptic defects associated with autism spectrum disorders.108,109 iPSC-derived neurons from patients with autism spectrum disorders are a promising means to assess the role of axon guidance proteins in the changes in neuronal morphology and connectivity that are found in patients. Furthermore, these cultures will not only help understand the cellular defects mediated by axon guidance protein dysfunction, but can also be used to functionally assess specific mutations at the biochemical and cellular levels. Although still in its infancy, the study of axon guidance proteins in neurological disease is an area of great potential. Although the pathogenic role of axon guidance proteins needs to be firmly established for most neurological disorders, in some disorders axon guidance proteins and their receptors have already been validated as therapeutic targets in experimental disease models. The signalling cues and cellular pathways that function downstream of axon guidance proteins and receptors could be used as targets for treatments. For example, investigators have postulated that defects in local protein synthesis contribute to the cause of neurodevelopmental disorders.110 mRNAs can be targeted to axons and dendrites, which enables rapid changes in the local proteome through local translation.111 Local translation has an important role in axon guidance, and axon guidance proteins such as Sema3A and Netrin-1 are known to regulate local protein synthesis to exert their effects on growing axons.111 Dysregulation of axon guidance proteins might lead to changed protein synthesis and thereby to disease. Another important downstream target of axon guidance proteins is the cytoskeleton. Several genes that encode proteins that regulate cytoskeletal dynamics have been linked to neurological disease. For example, mutations in CHN1, which encodes α2-Chimaerin, perturb axon guidance in the oculomotor system and lead to the eye movement disorder Duane retraction syndrome.112 With use of the CHN1 mutations as a starting point, cell culture and mouse studies have shown that the Semaphorins Sema3A and Sema3C act upstream of α2-Chimaerin and use this signalling cue to control the cytoskeleton and oculomotor axon guidance.112 This work not only identifies Sema3 signalling proteins as therapeutic targets in Duane retraction syndrome but also exemplifies how an intracellular signalling cue can be used to identify other downstream and upstream components of a specific disease pathway. In future studies, it will be interesting to establish whether the genes encoding Sema3s, their receptors, and downstream signalling cues carry mutations in patients with Duane retraction syndrome. 12

Another class of molecules that are promising therapeutic targets for disorders related to axon guidance proteins are microRNAs. For example, pathway analysis of the targets of microRNAs dysregulated in experimental temporal lobe epilepsy identifies the axon guidance pathway as one of the most frequently targeted pathways.113 This not only confirms the importance of axon guidance proteins in epilepsy but also raises the possibility that methods to manipulate microRNAs might be used to counteract epilepsy-associated pathological changes in neuronal connectivity caused by the abnormal expression of axon guidance proteins. Although much remains to be learned about the role and mechanism of action of axon guidance proteins in neurological disorders, prospects are good for the development of novel treatments by use of the axon guidance pathway as a therapeutic target. Contributors EVYB, SB and RJP reviewed the literature and wrote the manuscript. EVYB and SB prepared the figures and table, with input from RJP. Declaration of interests We declare no competing interests. Acknowledgments Our work was supported by the Dutch Epilepsy Fund, the Prinses Beatrix Fund, the International Parkinson Fund, the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7) 2007–2013 under REA grant agreement number 289581 (NPlast), and the FP7-HEALTH-2013-INNOVATION-1 Collaborative project Epi-miRNA. Our work was partly performed within the framework of CTMM (to RJP), the Center for Translational Molecular Medicine, project EMINENCE (01C-204). References 1 Kolodkin AL, Tessier-Lavigne M. Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb Perspect Biol 2011; 3: pii: a001727 2 Pasterkamp RJ, Kolodkin AL. SnapShot: Axon Guidance. Cell 2013; 153: 494–494e1–2. 3 Izzi L, Charron F. Midline axon guidance and human genetic disorders. Clin Genet 2011; 80: 226–34. 4 Nugent AA, Kolpak AL, Engle EC. Human disorders of axon guidance. Curr Opin Neurobiol 2012; 22: 837–43. 5 Geschwind DH, Levitt P. Autism spectrum disorders: developmental disconnection syndromes. Curr Opin Neurobiol 2007; 17: 103–11. 6 Amaral DG, Schumann CM, Nordahl CW. Neuroanatomy of autism. Trends Neurosci 2008; 31: 137–45. 7 McFadden K, Minshew NJ. Evidence for dysregulation of axonal growth and guidance in the etiology of ASD. Front Hum Neurosci 2013; 7: 671. 8 Lin L, Lesnick TG, Maraganore DM, Isacson O. Axon guidance and synaptic maintenance: preclinical markers for neurodegenerative disease and therapeutics. Trends Neurosci 2009; 32: 142–49. 9 Schmidt ERE, Pasterkamp RJ, van den Berg LH. Axon guidance proteins: novel therapeutic targets for ALS? Prog Neurobiol 2009; 88: 286–301. 10 Kubo T, Tokita S, Yamashita T. Repulsive guidance molecule-A and demyelination: implications for multiple sclerosis. J Neuroimmune Pharmacol 2012; 7: 524–28. 11 Kumanogoh A, Kikutani H. Immunological functions of the neuropilins and plexins as receptors for semaphorins. Nat Rev Immunol 2013; 13: 802–14. 12 Pasterkamp RJ. Getting neural circuits into shape with semaphorins. Nat Rev Neurosci 2012; 13: 605–18. 13 Lai Wing Sun K, Correia JP, Kennedy TE. Netrins: versatile extracellular cues with diverse functions. Development 2011; 138: 2153–69. Published online March 11, 2015


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Axon guidance proteins in neurological disorders.

Many neurological disorders are characterised by structural changes in neuronal connections, ranging from presymptomatic synaptic changes to the loss ...
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