Crmps: Critical Molecules for Neurite Morphogenesis and Neuropsychiatric Diseases

REVIEW CRMPs: critical molecules for neurite morphogenesis and neuropsychiatric diseases

TT Quach1,2, J Honnorat1,3,4, PE Kolattukudy5, R Khanna6 and AM Duchemin7

Neuronal polarity and spatial rearrangement of neuronal processes are central to the development of all mature nervous systems. Recent studies have highlighted the dynamic expression of Collapsin-Response-Mediator Proteins (CRMPs) in neuronal dendritic/ axonal compartments, described their interaction with cytoskeleton proteins, identiﬁed their ability to activate L- and N-type voltage-gated calcium channels (VGCCs) and delineated their crucial role as signaling molecules essential for neuron differentiation and neural network development and maintenance. In addition, evidence obtained from genome-wide/genetic linkage/proteomic/ translational approaches revealed that CRMP expression is altered in human pathologies including mental (schizophrenia and mood disorders) and neurological (Alzheimer’s, prion encephalopathy, epilepsy and others) disorders. Changes in CRMPs levels have been observed after psychotropic treatments, and disrupting CRMP2 binding to calcium channels blocked neuropathic pain. These observations, altogether with those obtained from genetically modiﬁed mice targeting individual CRMPs and RNA interference approaches, pave the way for considering CRMPs as potential early disease markers and modulation of their activity as therapeutic strategy for disorders associated with neurite abnormalities.

Molecular Psychiatry (2015) 20, 1037–1045; doi:10.1038/mp.2015.77; published online 16 June 2015

The ability of the nervous system to process continuously maintenance of axodendritic arborization and discuss their changing information from the internal and external environ- potential involvement in human nervous system diseases. ments requires the development of precise networks of neurons and structural plasticity of their dendrites and axons. Considering the intricacies of specific axodendritic connectivity, it is surprising CRMPS: CHARACTERISTICS AND EXPRESSION that neurons can undergo such precise structural reorganization in CRMPs are five homologous cytosolic phosphoproteins.6–8 CRMP1, response to environmental change. However, evidence indicates 2, 3 and 4 display 75% homology with each other,9–11 whereas that far from being static structures, axons and dendrites are the phylogenetically divergent CRMP5/CRAM shares only 50% plastic and this plasticity, which is part of a lifelong process, is homology.12 CRMP1, 2 and 4 have two distinct alternatively mediated by various proteins such as neurotrophic/transcription spliced isoforms in their N termini,13 extending the number of factors, neurohormones, guidance cues, membrane receptors, active molecules in this family. All CRMPs assemble in heterophilic voltage-gated calcium channels (VGCCs) and involves rearrange- oligomers in vivo, bind to tubulin14 and are phosphorylated by ment of cytoskeletal proteins that support the structural changes.1 various kinases that regulate their activity.15 They act as signaling However, despite the rich information on mechanisms underlying phosphoproteins modulating cytoskeletal organization and neuronal differentiation, axonogenesis and dendritogenesis, the regulating neurite formation and retraction. Site-specific phos- key molecules involved in the initiation of neurites through phorylation of CRMPs determines their affinity for binding to cyto- formation of lamellopodia/filopodia, specification and maturation skeletal proteins and their neurite-promoting/collapsing activities. – of neurites, and genesis of spines still remain elusive.2 3 Because CRMP1 appears to be a target of RhoA signaling.16 The Sema3A17 such a large fraction of the genome is expressed in the brain and and reelin18 signaling pathways are regulated by the phosphor- our knowledge of mechanisms involved in neuronal connectivity ylation of CRMP1 by Cdk5 at Thr-509 and Ser-522, or by Fyn at is still in its infancy, it has been postulated that more mole- Tyr-504 that lowers its binding affinity to tubulin. Similarly, non- cular cues and pathways specialized for axonal/dendritic/spine phosphorylated CRMP2 binds strongly to tubulin leading to genesis and synaptic network formation remain to be iden- microtubule formation, whereas its phosphorylation by Rho kinase tified. Consistent with this notion, Collapsin-Response-Mediator suppresses its binding to tubulin and Numb.14 Two other kinases Proteins (CRMPs) can now be considered as building blocks for working in tandem, Cdk5 and GSK-3β, also control the interaction neuronal differentiation and neurite network organization and of CRMP2 with tubulin. In addition, CRMP2 regulates the stability remodeling.4–5 In this review, we outline the current evidence of actin filaments.19 CRMP3 is phosphorylated by Fyn in vitro15 but showing that CRMPs are important in the establishment and is unique among CRMPs in that the three GSK-3β phosphorylation

1Lyon Neuroscience Research Center, INSERM U1028/CNRS UMR 5292, F-69372 Lyon, France; 2Department of Neuroscience, The Ohio State University, Columbus, OH, USA; 3French Reference Center on Paraneoplastic Neurological Syndrome, Hospices Civils de Lyon, Hôpital Neurologique, Neurologie B, Bron, France; 4Université de Lyon, Université Claude Bernard Lyon 1, F-69372 Lyon, France; 5Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, USA; 6Department of Pharmacology and Neuroscience, Graduate Interdisciplinary Program, College of Medicine, The University of Arizona, Tucson, AZ, USA and 7Department of Psychiatry, College of Medicine, The Ohio State University, Columbus, OH, USA. Correspondence: Dr AM Duchemin, Department of Psychiatry, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. E-mail: [email protected] Received 7 November 2014; revised 29 April 2015; accepted 8 May 2015; published online 16 June 2015 CRMPs in neuron development and diseases TT Quach et al 1038

Table 1. Summary of relative CRMP expression pattern in mouse nervous system

Cortex Hippocampus Dentate gyrus Striatum Cerebellum Pons DRG

CRMP1 ePN + + + ? +++ ? ? Adult ++ +++ ++ ? +++ ? ? CRMP2 ePN ++ ++ ++ ++ ++ ? ++ Adult ++ +++ ++ + ++ ? + CRMP3 ePN − +++ +++ − +++1 ++ Adult − +++ +++ −−++1 − CRMP4 ePN ++ +++ +++ + + +2 ? Adult − ++−−−+ CRMP5 ePN + + + ? + ? ? Adult + ++ ++ + ++ ? ? Abbreviations: DRG, dorsal root ganglia; ePN, early postnatal. (1), inferior olive complex and reticular formation; (2), pontine nuclei. Data from refs 9,12,13,23,29,31,32,44,46,48,127–129; +++, high level; ++, moderate level; +, low level; − , undetectable signal; ?, not determined.

sites identified in other CRMPs are not conserved in this molecule. activation of L- and N-type VGCCs by CRMP3 provide strong CRMP4, which binds to F-actin and regulates F-actin bundling,20 is support for an inductive role of CRMP3 in neurite initiation and also a physiological partner of GSK-3β.21 Of note, GSK-3β is a dendritic development and plasticity, at least partly via Ca2+ ubiquitous protein kinase having a pivotal role in neurodevelop- influx29 (Figure 1d). In contrast, CRMP5 depletion by RNA ment and is suspected to be involved in neuropsychiatric interference enhances the length and number of neurites, disorders such as schizophrenia,22 bipolar disorders23 and whereas its overexpression induces mitophagy and reduces Alzheimer’s disease (AD).24 CRMP5 is a substrate for Cdk525 and dendritic length. Further, overexpression of mutated-Threo-516 26 is tyrosine-phosphorylated by Fes/Fps and TrkB. In view of these CRMP5 (ref. 34) and/or its truncated dominant-negative isoform observations, we can conclude that, depending on the character- lacking the tubulin-binding domain35 has no effect on dendrite istics of their phosphorylation status, CRMPs may associate with formation or length, demonstrating the critical role of the tubulin- 27 various partners and signaling pathways, some of which may binding domain in the neurite outgrowth inhibition induced by exclude direct interaction between them in different brain areas. CRMP5. The interaction of CRMP5 with tubulin has important 28 29 30 In addition, CRMP2, CRMP3 and CRMP4 have been shown to ramifications for the function of CRMP2. Consistent with the modulate calcium flux, another mechanism of neurite regulation. 10 31 notion that the activities of CRMPs are inter-related, dendritic According to in situ hybridization, immunostaining and arborization induced by CRMP2 is considerably enhanced by 32 fi gene replacement analyses, all ve CRMPs are highly expressed CRMP5 deficiency.36 There is also evidence that CRMP4 regulates in the brain during early postnatal development and continue to dendritic outgrowth: CRMP4–short hairpin RNA-transfected be present in specific regions into adulthood, especially in areas and CRMP4 − / − cultured hippocampal neurons show increased with extensive neuronal plasticity (Table 1). They are found in dendritic branching without significant changes in primary axons and dendrites of the neurons where they contribute to dendrite number.37 This effect is partly dependent on Sema3A specific signaling mechanisms involved in the regulation of axonal signaling through F-actin bundling.20 Phosphorylated CRMP1 and and dendritic development and maintenance. CRMP2 are localized in the dendrites of cortical neurons where they regulate dendritic branch trajectories.38 Mice with abnormal phosphorylation of CRMP1 and CRMP2 present a curling dendrite CRMPS INFLUENCE NEURITE INITIATION AND DENDRITIC 38 OUTGROWTH phenotype, suggesting that these two molecules are critical for normal dendrite patterning in cortical neurons. The roles of molecules critical for dendritic specification have been examined extensively in cultured primary neurons. In vitro studies examining the selective contribution of CRMPs to dendritic CRMPS AND AXON SPECIFICATION development revealed their differential roles. In cultured hippo- It has been assumed that axon–dendrite polarity involves campal neurons, CRMP3 is essential, not only for dendrite exten- polarized transport of various cargoes, actin instability, complex sion, a late-stage event in the differentiation of neurons, but also microtubule orientation and distinct repertoires of extracellular in lamellopodia formation, the early stage of neurite initiation matrix/structural/signaling proteins. Some proteins are specific for (Supplementary Information, SI). Although neurons from control either dendritic or axonal specification: for example, down- wild type and CRMP3+/ − mice polarize and grow normally, all regulation of myelin-associated protein-2 inhibits dendrite forma- neurons lacking CRMP3, identified by their expression of the LacZ- tion, whereas downregulation of Tau1 inhibits axon formation. reporter gene (SI-A), from CRMP3 − / − littermates do not initiate However, several molecules including CRMP2 can affect both neurite formation.29 They remain spherical, even after 10 days dendrites38 and axons.39 In hippocampal neurons, overexpression in vitro. This defect in neuritogenesis is largely due to a failure to of CRMP2 induces supernumerary axons, and increases axonal evolve from stage 1 to stage 2, defined as neurite initiation. The branching, whereas its inhibition with a dominant-negative first step in neuronal polarization requires the establishment of protein suppresses the formation of the primary axon.39 Interest- lamellopodia, considered as the principal means of initiating the ingly, CRMP2 can convert established dendrites to axon. It is formation of primary neurites. Consistent with this finding, CRMP3 involved in the Sra-1/WAVE/kinesin cargo complex and is required overexpression promotes lamellopodia formation and significantly for brain derived neurotrophic factor (BDNF) and neurotrophin 3 increases both the number and length of dendrites (Figure 1). In (NT3)-induced axon outgrowth/branching.19,40 How might CRMP2 cortical neurons, Ena/VASP deletion also prevents neurite initiation fulfill this function? Axon elongation is achieved through micro- but by inhibiting filopodia formation,33 the second step in tubule assembly and extension, actin reorganization and mem- establishing the primary neurites. The correlation between levels brane genesis at the leading edge of the growth cone. CRMP2 of CRMP3 expression, dendrite/spine morphogenesis and the may not only enhance microtubule polymerization but may also

Molecular Psychiatry (2015), 1037 – 1045 © 2015 Macmillan Publishers Limited CRMPs in neuron development and diseases TT Quach et al 1039 complex formation is critical for outgrowth inhibition via an actin-dependent process.30 Ectopic expression of the short isoform of CRMP4 enhances axonal extension, whereas its dephosphorylated long isoform mediates the inhibition of myelin-associated inhibitor- induced axonal outgrowth illustrating that the long isoform can serve as short isoform antagonist. The role of CRMP1 in axonogenesis was demonstrated by the abolition of axonal extension induced by NT3 in chick dorsal root ganglia neurons using speciﬁc antibodies or CRMP1-mutant proteins.43 In CRMP1 − / − mice, axonal lengths in cerebellar explant cultures are not altered,44 suggesting selective functional activity of CRMP1 depending on cell types and/or species. Finally, despite their role in dendritogenesis, overexpression or deletion of CRMP3 in cultured hippocampal CA1 neurons29 and deletion of CRMP5 in cerebellar explant cultures36 yield no apparent alteration of axon morphology, suggesting that some CRMPs may affect only dendrites.

PHENOTYPES OF CRMP-DEFICIENT MICE Although the cultured neuron is a very valuable model, one must be cautious when extrapolating results to in vivo situations. Polarity and asymmetry can be altered by disruption of cell–cell and cell–extracellular matrix interactions. Moreover, not all neurons select an axon from primary neurites as in the case of hippocampal neurons.45 In the living animal, the difference in axon-versus-dendrite genesis may be linked to the presence of extracellular cues affecting the immature neuron.33 Gene- targeting inactivation has enabled elucidation of unique functions of individual CRMPs in different cell types in vivo, although adaptive compensation by related CRMP members must be kept in mind. Figure 1. Effects of CRMP3 on dendrites of hippocampal neurons. ● CRMP1 − / − mice display abnormal granule cell migration and (a) Overexpression of CRMP3 increases dendritic arborization. 45 Dissociated 1 d.i.v. hippocampal neurons were transfected with apoptosis in the cerebellum, and abnormal GAP43/PSD95/ Flag-CRMP3 or Flag-vector, fixed at 4 days in vitro (div) and immuno- myelin-associated protein-2/Golgi staining in the CA1 area of stained for Flag (red) and dendritic marker myelin-associated hippocampus consistent with a structural alteration of protein-2 (MAP2; green). (A) Representative untransfected neuron. neurites.46 Behaviorally, CRMP1 − / − mice exhibit abnormalities (B) Neurons expressing control Flag-vector. (C) Neurons expressing in long-term potentiation (sSI), motor activity, emotional Flag-CRMP3. Merged images showed transfected neurons in yellow behavior, long-term memory retention and prepulse inhibition (bottom panels). Some Flag-CRMP3-expressing neurons display an (sSI); the latter is rescued by treatment with chlorpromazine, an increase of more than three times in the number of dendrites. (b)A proposed model for the regulation of dendrite morphology by antipsychotic drug. These observations raise the possibility that CRMP3. (1) Combinatorial stimulation of CRMP3 gene expression or CRMP1 − / − -associated dendrite and spine dysfunction may activity by intrinsic and extrinsic factors. (2) The localization and involve deficits in information processing. Interestingly, in vivo distribution of FL-CRMP3-positive puncta in dendrites suggests that microdialysis revealed an increased release of dopamine in the —similar to CRMP2—CRMP3 could be associated with vesicles or prefrontal cortex, which might contribute to the observed large carrier protein complexes, and actively transported to hyperactivity of CRMP1 − / − mice.47 – dendrites. (3 4) Structural and biochemical studies support the ● CRMP2 − / − mice: our attempt to target the CRMP2 gene was idea that CRMP3 activity might be regulated by phosphorylation or unsuccessful (Quach et al., unpublished observation) suggesting other post-translational modifications induced by (7) neurotrophic factors/guidance cues. Activated CRMP3 binds to different partners a critical role of CRMP2 in embryonic development. and serve as adaptor to a variety of signaling pathways including ● Analysis of CRMP3 − / − mice supports a role for CRMP3 in Ca2+ influx (CaV). CRMP3 contributes to (8) neurite initiation, (5) hippocampal dendritic organization and function. CRMP3 − / − dendrite arborization and (6) spine genesis. These effects may mice display behavioral alteration concordant with hippo- involve lamellopodia formation. Reproduced from Quach et al.29 campal functional impairment such as a decrease in long- term potentiation48 and prepulse inhibition.49 They present abnormal hippocampal dendrites characterized by decreased modulate vesicle/membrane trafficking/remodeling through inter- dendritic length and number of branching points, abnormal action with its partners, depending on specific post-translational undulation of apical primary dendrites and abnormal dendritic 48 modifications19,41–42 and secreted trophic factors. In hippocampal spine structure. The abnormal undulation, also present in L1 neurons, BDNF and NT3 induce axonal growth and branching knockout mice,50 is most likely due to altered signaling of activity by inhibiting CRMP2 phosphorylation by GSK-3β.39–40 CRMP3-dependent guidepost proteins directing point-to-point Conversely, Sema-3A induces axonal retraction by increasing navigation of growth cones on the spatial array. L1 is the co- CRMP2 phosphorylation by GSK-3β.15 Short interfering RNA- receptor of the Neuropilin1 receptor complex for Sema 3A. mediated knockdown of CRMP4 in the dorsal root ganglia Intriguingly, while dendrites are altered but remain present attenuates neurite outgrowth inhibition induced by myelin in vivo, the absence of neurites is the common phenotype of substrate, identifying CRMP4 as an inhibitor of axonal CRMP3 − / − cells in vitro.29 The apparent difference in cellular outgrowth.27 The long variant CRMP4 increases Ca2+ current in phenotypes obtained from the identical genotype may be whole-cell voltage clamp experiments and the CRMP4-RhoA related to functional redundancy between proteins and partial

© 2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 1037 – 1045 CRMPs in neuron development and diseases TT Quach et al 1040 compensatory mechanisms of repair in vivo.29 Guidance cues/ involved in neuronal migration and neurite/synapse formation. extracellular matrix /neurotrophic factors that possibly activate Recent studies of gene copy number variants and exome other pathways leading to neurite formation in CRMP3 − / − sequencing of single-nucleotide polymorphisms (SNPs) impli- neurons may be absent in cultured cells. cate genes modulating cytoskeleton proteins, NMDA receptors ● In CRMP4 − / − mice, the distance from the soma to the first and VGCCs, converging on pathways regulating synaptic bifurcation point in the apical dendrites of CA1 pyramidal plasticity.55 It is not surprising that CRMP1, also called neurons is significantly decreased, confirming an inhibitory role dihydropyriminidase-like 1 (DPYSL1), which interacts with reelin of CRMP4 on dendrite bifurcation and branching.37 Otherwise, and DISC1,18 and CRMP2 (DPYSL2), which affects not only 2+ these mice have no observable changes in the CNS structure. neurites but also neurotransmitter release and modulates Ca 28 ● In CRMP5 − / − mice, anti-calbindin immunostaining revealed fluxes through activation of NMDA receptors, have been altered cell body sizes and dendrites.26 In addition, the dendritic included in the list of schizophrenia susceptibility genes. CRMP1 branching induced by BDNF is markedly attenuated in these was identified as altered protein in a search for schizophrenia- 56 animals. Significant deficiencies in long-term depression (sSI) specific epitopes from patients’ post-mortem brain fractions. and abnormal limb-clasping reflexes are consistent with A genetic analysis corroborated the association of anhedonia 56 abnormal central synaptic function. These mice also develop with the CRMP1 gene locus in a Finnish population cohort. abnormalities in motor conduction velocity, polyaxonal Furthermore, CRMP1 immunoreactivity is increased in lympho- ensheathment and non-myelinating Schwann cell structure, blastoid cells derived from patients diagnosed with schizophrenia, suggesting that CRMP1 may be a potential biomarker and mimic a paraneoplastic neurological disorder syndrome 56 found to be associated with anti-CRMP5 auto-antibodies.51 of the disease. However, the precise mechanisms by which endogenous CRMP1 disrupts brain circuitry in patients, and However, the mechanisms underlying the structural changes 48 are unknown. It is possible that signaling changes originate in how the deletion of its gene disrupts brain circuitry in rodents the axon and initiate dysfunction in glial cells, which in return ultimately leading to behavioral similarities found in compara- leads to axonopathy. Complementary to gene deletions, tive phenotypic analyses, such as enhanced mesocortical dopaminergic transmission and decrease in prepulse inhibition transgenic mice overexpressing CRMPs may enhance our (also present in CRMP3 − / − mice49), remain to be determined. understanding of the molecular pathways and mechanisms Multiple proteome-wide analyses have identified significant underlying human pathology. changes in levels of CRMP2 in the brain of schizophrenia patients.57–64 Corroborating these data, a more-than-6000-SNP- linkage scan of 831 pedigrees, confirmed by an independent HUMAN NERVOUS SYSTEM DISEASES: INSIGHTS FROM Ashkenazi case–control and a family-based association of CRMP-ALTERED LEVELS/ACTIVITY AND ANIMAL MODELS European-Caucasian pedigrees, found a schizophrenia susceptibility 1-Mb region on chromosome 8p21 that contains the CRMP2 gene.65 Previous multicenter genome-wide linkage ● 52 Schizophrenia is considered a neurodevelopmental disorder studies have consistently showed strong evidence implicating characterized by ventricular enlargement and decreased 8p21 susceptibility locus for schizophrenia66–68 and CRMP2 volume of the frontal cortex and hippocampus, with a reduction gene in particular.69 Recent findings of association of several in size of neuronal soma, shortened length of axons and functional gene variants of CRMP2/DPYSL2 with schizophrenia dendrites and loss of spines in the prefrontal cortex and suggest that alterations of CRMP2 transcription and translation 53–54 hippocampus. Altered prepulse inhibition is a common increase the risk for the disease.70 However, despite finding an neurophysiological endophenotype. Transcriptomic studies association of CRMP2 polymorphism with schizophrenia in argue that the molecular underpinnings of the disease are Japanese patients, Koide et al.71 did not replicate it in another even more varied than its symptomatic diversity, and that group. Explanations for the conflicting linkage findings may changes in the transcriptome can affect developmental come from various factors present in (a) the disorder itself (its processes. Once compensatory mechanisms are exceeded, multifactorial genetic–allelic difference, phenotypic hetero- alterations manifest as a specific behavioral phenotype geneity and possible epigenetic nature), (b) methods (popula- corresponding to the symptoms of the disease. Discovery of tion pedigree, gender, ethnicity and sample sizes) and (c) NRG1/Akt/dysbindin-1, and DISC-1 as susceptibility genes for technology (SNPs are different between studies: for example, schizophrenia, suggested an etiopathogenic role for molecules the SNPrs 12155555 that shows strong association to

Figure 2. Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex. (a) Normalized binding of CaV2.2 to 15-mer peptides (overlapping by 12 amino acids) encompassing full-length CRMP-2 overlaid with spinal cord lysates. The sequence of peptide 96, designated CBD3, is shown. (b) Left: representative differential interference contrast/fluorescence images showing robust penetration of fluorescein isothiocyanate-TAT-CBD3 into dorsal root ganglia (DRGs; arrowheads) but not other cells (arrows). Nuclei are stained with Hoechst dye in the bottom image. Right: representative current traces from a dorsal root ganglia (DRG) incubated for 15 min with TAT-Scramble (10 μM; black) or TAT-CBD3 (10 μM; cyan) in response to the voltage steps illustrated at the top. (c) Representative current clamp recordings from small-to-medium (430440 μm) diameter lumbar four to five DRG neurons demonstrating that application of 10 μM CBD3 (cyan) reduces the number of elicited action potentials; grouped data are shown in the bar graphs on the right. (d) Representative traces of spontaneous EPSCs (sEPSCs) in lamina II neurons in spinal cord slices before treatment (left) or after application of 10-μM TAT-Scramble (middle) or 10-μM TAT-CBD3 (right). Bottom, enlarged traces. Voltage-clamp recordings (holding voltage, –70 mV) were used to record synaptic responses. Right: ratio of sEPSC frequency and amplitude. *Po0.05 compared with baseline. (e) The effect of peptides on number of flinches on formalin-induced phase 1 (0–10 min) and phase 2 (15–60 min). (f) Paw withdrawal threshold (PWT; in mN) of rats injected once with the antiretroviral drug ddC and treated with TAT peptides at 7 days following ddC administration. *Po0.05 versus ddC or TAT-Scramble (analysis of variance (ANOVA) with Student–Newman–Keuls post hoc test). (g) Visualization of FITC fluorescence in DRGs, isolated 15 min after injection of FITC-TAT-CBD3 and immunolabeled with a NeuN-specific antibody (NeuN; red). Cells were also stained with Hoechst (blue), which labels cell nuclei. (h) Cartoon depicting the main hypothesis which proposes that uncoupling the CRMP-2-CaV2.2 interaction leads to a physiologically relevant decrease in Ca2+ current and neurotransmitter release and, in turn, suppresses persistent inflammatory and neuropathic hypersensitivity. Reproduced from Wang et al.115 and Brittain et al.121

Molecular Psychiatry (2015), 1037 – 1045 © 2015 Macmillan Publishers Limited CRMPs in neuron development and diseases TT Quach et al 1041 schizophrenia (P = 0.000063)63 was not tagged by Koide et al.71). of schizophrenia susceptibility genes inhibits the expression Levels of CRMP4 (DPYSL3) are decreased in the CA2/CA3 areas of CRMP4.73 CRMP4 is also a target for the NMDA-regulated of the hippocampus of schizophrenia patients,72 and these microRNA-132, which is downregulated in the prefrontal cortex changes may be under the controls of microRNAs altered in the of subjects with schizophrenia.74 Interestingly, CRMPs were disease. The overexpression of microRNA-137 coded by one identiﬁed in a proteomic proﬁle of rat cerebral cortex proteins

© 2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 1037 – 1045 CRMPs in neuron development and diseases TT Quach et al 1042 changed after chronic administration of two antipsychotic disorder,94 supports common etiopathogenic mechanisms for drugs, clozapine and risperidone:75 ‘Alterations in CRMP2 and disease symptomatology. CRMP4 were the most spectacular consequences of treatment ● Alzheimer’s disease: Since various forms of memory require an with both drugs’ the authors reported. Increased CRMP2 levels intact hippocampus, it is not surprising that it is one of the in schizophrenia correlate with antipsychotic exposure.62 earliest and most vulnerable brain regions affected by the Protein levels in patients may depend on brain area, stage of disease.95 In fact, hippocampal synaptic dysfunction and the disease and psychotropic use. In addition, chronic treat- memory impairment in AD are recapitulated in AD transgenic ment of rats with phencyclidine, which can mimic schizo- models with neuritic senile plaques (β-amyloid) and neurofi- phrenia symptoms, downregulates CRMP2 and upregulates brillary tangles (NFTs), which support the neuropathological CRMP4 levels in the medial prefrontal cortex.76 Despite these concept of a ‘dendritic-dysconnectivity’ syndrome96 associated supportive observations, the primary extent to which CRMP with aberrant NMDA-R activation, repressor element 1-silencing expression is associated with schizophrenia awaits further transcription factor (REST) expression97 and calcium signaling confirmation. perturbation.98 Interestingly, increased phosphorylated ● Mood disorders: major depressive disorder (MDD) is associated CRMP2 has been detected in the soluble fraction of AD- with hippocampal atrophy characterized by decreased density affected human brain tissue and in NTFs.99 In transgenic mouse in dendrites and dendritic spines that can be reversed models, phosphorylated CRMP2 is increased in hippocampus by antidepressant treatments.77 The role of stress, and espe- well before plaque and tangle formation.100 Although the cially early-life trauma, leading to hippocampal atrophy is relationship between pathophysiological mechanisms and AD well documented in the development of depression. In an symptoms remains to be clarified, these findings position animal model of early-life stress, the levels of CRMP5—an phosphorylated CRMP2 as a potential biomarker for early AD, inhibitor of dendritic growth—are increased while tubulin and especially since it seems specific to this type of dementia.101 In microtubule-associated protein are decreased.78 In MDD, the an animal model of AD, suppression of CRMP2 phosphorylation structural dendrite alterations associated with the depressive is associated with amelioration of β-amyloid-induced cognitive symptoms seem related to a deficiency in neurotrophic dysfunction and hippocampal axon degeneration.102 factors.79 Levels of the brain and serum BDNF are decreased ● Human prion diseases, which include sporadic/familial Creutz- in patients with depression.80–81 Furthermore, it has been feld–Jacob disease, are disorders of protein conformation in shown that (1) BDNF may be involved in the MDD gene– which PrPc, the normal cellular conformer, is converted to the environment interaction model82 and (2) BDNF+/ − knockout83 abnormal protease-resistant PrPSc.103-104, In animal models, a and BDNF Val66/Met knock-in84 mice display reduction in specific increase in CRMP4 expression105 and dendritic neurite length, altered dendritic spines and depression-like degeneration106 are the two earliest morphological features, behaviors. The role of CRMPs in BDNF effects is supported by detectable in mice before clinical symptoms are evident. In the the facts that (1) BDNF blocks phosphorylation of CRMP2 via the late stage of the disease, the amount of CRMP2-ΔC that lacks PI3-kinase/AKt/GSK3-β pathway40 and (2) knockdown of the sequence from Ser-518 to the C terminus is increased CRMP2 causes a marked inhibition of BDNF-induced axon significantly.107 In cultured neurons, it was found that PrP106–126 elongation and branching,40 indicating that CRMP2 is necessary induces dystrophic changes in dendrites, an effect prevented for at least some of BDNF neurotrophic activities. In addition, by overexpression of CRMP3.108 It is worthwhile to note that in CRMP2 levels are decreased in the brain of patients with humans, the hippocampus, where CRMP3 is highly expressed, is depression.59 As antidepressants release neurotrophic factors protected from the extensive neuronal loss of the spongiform and/or modulate protein kinases such as GSK-3β,85 their degeneration until the final stage of disease.108 activities on dendrites and spines may be mediated in part by ● Epilepsy: epilepsy can be considered as a disorder of ion channel CRMPs. Indeed, proteomic analysis shows that chronic treat- dysfunction and abnormal brain networks.109 The pathogenic ment with two antidepressants, venlafaxine and fluoxetine, role of the genetic mutations of CDKL5, SYN1, ARHGEF9, DCX increases the level of CRMP2 in hippocampus by 4.0 and 2.6 and MeCP2 involves neuronal migration, neurite pruning, protein times, respectively.86 Tianeptine, an atypical antidepressant transporters, synaptogenesis, neurotransmitter release and func- with neurotrophic activity, also increases CRMP2 expression.87 tions of receptors. In patients with temporal lobe epilepsy, there The rapid antidepressant ketamine, an NMDA receptor antago- is a reduction in fiber density in the limbic structure110 and nist, also requires release of BDNF and inhibition of GSK3β thickness of the entorhinal cortex111 with abnormal global suggesting a common final pathway for antidepressant network efficiency.112 CRMP1 expression is decreased in the activity.88 Together, these observations suggest that CRMP2 temporal neocortex of patients with intractable temporal lobe may be involved in MDD and/or its treatment. In bipolar epilepsy113 as well as in pilocarpine-induced animal model of disorder, CRMP2 protein levels are decreased in the frontal epilepsy.113 Proteomic analysis of hippocampal tissue from cortex and CA2/CA3 areas of the hippocampus.59,70 CRMP4 is patients with mesial temporal lobe epilepsy revealed a drastic also decreased in the hippocampus.72 Genome-wide associa- decrease in CRMP2 protein.114 It is tempting to suggest that tion studies have detected several genetic markers for bipolar abnormal expression of the two proteins impairs neurite disorder, including CACNA1C,89 that encodes a subunit of the outgrowth and axonal pathfinding, contributing to the abnormal L-type voltage-dependent calcium channel shown to be structural connectivity found in epilepsy, although whether they associated with the dendritic arborization activity of CRMP3 in are a cause or an effect of seizures remained to be established. the hippocampus.29 The mood stabilizer lithium is a potent The anti-epileptic drug lacosamide binds directly to CRMP2 and GSK-3β inhibitor that inhibits CRMP2 phosphorylation in this property is proposed as one of its mechanisms of action.115 cultured rat cortical neurons.90 Lithium neurite elongation and ● Neuropathic pain (NP): sprouting after nerve injury probably branching activities may result in part from changes in CRMP2 induced by neurotrophic factors, for example, nerve growth phosphorylation. In bipolar patients, lithium treatment factor (NGF) for the sympathetic system,116–117 and maladaptive increases total grey matter and hippocampal volumes and spine remodeling, for example, in diabetic neuropathy,118 may improves white matter integrity altered by the disease,91–93 explain the neuron hyperexcitability and decrease in pain suggesting structural changes of neurites. Evidence of shared threshold characteristics of NP. Although N-type VGCCs (CaV2.2) genetic variations between psychiatric disorders, with high are genetically119 and clinically120 validated targets for pain correlation between schizophrenia and bipolar disorder, and management, the CaV2.2-selective blockers have untoward side moderate correlation between MDD and schizophrenia/bipolar effects. Interestingly, a CRMP-2-derived peptide (tat-CBD3;

Molecular Psychiatry (2015), 1037 – 1045 © 2015 Macmillan Publishers Limited CRMPs in neuron development and diseases TT Quach et al 1043 Figure 2; Supplementary Information, SI-B) that disrupts the 10 Wang LH, Strittmatter SM. A family of rat CRMP genes is differentially expressed CaV2.2/CRMP2 interaction proved to be antinociceptive in in the nervous system. J Neurosci 1996; 16: 6197–6207. animal models of inflammation and NP.121–124 Adeno- 11 Byk T, Ozon S, Sobel A. The Ulip family phosphoproteins: common and specific 254 – associated virus-encoded CBD3 delivered to rat peripheral properties. Eur J Biochem 1998; :14 24. 4 12 Inatome R, Tsujimura T, Hitomi T, Mitsui N, Hermann P, Kuroda S et al. Identifi- sensory neurons provides sustained relief ( 6 weeks) of NP. The cation of CRAM, a novel unc-33 gene family protein that associates with CRMP3 positive effect and relative lack of toxicity of the adeno- and protein tyrosine kinase(s) in the developing rat brain. J Biol Chem 2000; 275: associated virus-targeted expression of CBD3 peptide in this 27291–27302. animal model provide evidence of therapeutic promise for this 13 Quinn CC, Chen E, Kinjo TG, Bell AW, Eliott RC, McPherson PS et al. TUC-4b, type of approach to treat chronic NP125 and other disorders with a novel TUC family variant, regulates neurite outgrowth and associates with similar pathophysiological mechanisms. vesicles in the growth cone. J Neurosci 2003; 23: 2815–2823. 14 Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimara T, Shiromizu T et al. CRMP2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol CONCLUDING REMARKS 2002; 4: 583–591. There is no doubt that CRMPs have important roles in nervous 15 Uchida Y, Ohshima T, Yamashita N, Ogawara M, Sasaki Y, Nakamura F et al. system development and disease. They are intracellular mediators Sema3A signaling mediated by Fyn-dependent tyrosine phosphorylation of CRMP2 at tyrosine 32. J Biol Chem 2009; 284: 27393–27401. of neurotrophic factors/guidance cues regulating neurite structure/ 16 Leung T, Ng Y, Cheong A, Ng CH, Tan L, Hall C et al. P80 ROK alpha binding spine formation and are essential for dendrite patterning and protein is a novel variant of CRMP1 which associates with CRMP2 and modulates directional axonal pathfinding. The array of activities provided by RhoA-induced neuronal morphology. FEBS Lett 2002; 523: 445–449. the various members of the family depending on their amino-acid 17 Yamashita N, Morita A, Uchida Y, Nakamura F, Usui H, Ohshima T et al. sequences, their isoforms and their phosphorylation status encom- Regulation of spine development by semaphorin 3A through cyclin-dependent pass a variety of interrelated mechanisms for regulating dendrite kinase 5 phosphorylation of collapsin response mediator protein 1. J Neurosci 14 – and axon growth and retraction. Altogether, these observations 2007; : 12546 12554. 18 Yamashita N, Uchida Y, Ohshima T, Hira S, Nakamura F, Taniguchi M et al. CRMP1 indicate that CRMPs are important in maintaining the constantly mediates reelin signaling in cortical neuronal migration. J Neurosci 2006; 26: remodeling neuronal network that supports neuronal function. As 13357–13362. such, their involvement in neuropsychiatric diseases is not 19 Kawano Y, Yoshimura T, Tsuboi D, Kawabata S, Kaneko-Kawano T, Shirataki H surprising. Now that their involvement has been demonstrated et al. CRMP2 is involved in kinesin-dependent transport of the Sra-1/WAVE1 and is supported by data from animal models, and genetic linkage complex and axon formation. Mol Cell Biol 2005; 25:9920–9935. scanning, we expect more studies will focus on their role in the 20 Rosslenbroich V, Dai L, Baader SL, Gieselmann V, Kappler J. Collapsin response 310 – pathophysiology of these diseases. In a parallel effort, evidence mediator protein-4 regulates F-actin bundling. Exp Cell Res 2005; :434 444. 21 Cole AR, Knebel A, Morrice NA, Robertson LA, Irving AJ, Connolly CN et al. GSK-3 from pharmacological studies shows that CRMP activities are phosphorylation of the Alzheimer epitope within collapsin response mediator 75,86–87,90 modulated by various psychotropic drugs and suggests proteins regulates axon elongation in primary neurons. J Biol Chem 2004; 279: that they are compelling intracytoplasmic targets. Drug discovery 50176–50180. technologies targeting the ‘CRMP-activity/disease-change’ equation 22 Cole A. GSK-3 substrates in mood disorders and schizophrenia. FEBS J 2013; 280: are currently explored for neurological diseases,99,125,126 and may 5213–5227. bring new therapeutic interventions for psychiatric diseases as well. 23 Brennand JK, Simone A, Tran N, Gage FH. Modeling psychiatric disorders at the cellular and network levels. 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