Dpysl2 (CRMP2) and Dpysl3 (CRMP4) phosphorylation by Cdk5 and DYRK2 is required for proper positioning of Rohon-Beard neurons and neural crest cells during neurulation in zebrafish

Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos.     

Position fine-tuning of caudal primary motoneurons in the zebrafish spinal cord

In zebrafish embryos, each myotome is typically innervated by three primary motoneurons (PMNs): the caudal primary (CaP), middle primary (MiP) and rostral primary (RoP). PMN axons first exit the spinal cord through a single exit point located at the midpoint of the overlying somite, which is formed beneath the CaP cell body and is pioneered by the CaP axon. However, the placement of CaP cell bodies with respect to corresponding somites is poorly understood. Here, we determined the early events in CaP cell positioning using neuropilin 1a (nrp1a):gfp transgenic embryos in which CaPs were specifically labeled with GFP. CaP cell bodies first exhibit an irregular pattern in presence of newly formed corresponding somites and then migrate to achieve their proper positions by axonogenesis stages. CaPs are generated in excess compared with the number of somites, and two CaPs often overlap at the same position through this process. Next, we showed that CaP cell bodies remain in the initial irregular positions after knockdown of Neuropilin1a, a component of the class III semaphorin receptor. Irregular CaP position frequently results in aberrant double exit points of motor axons, and secondary motor axons form aberrant exit points following CaP axons. Its expression pattern suggests that sema3ab regulates the CaP position. Indeed, irregular CaP positions and exit points are induced by Sema3ab knockdown, whose ectopic expression can alter the position of CaP cell bodies. Results suggest that Semaphorin-Neuropilin signaling plays an important role in position fine-tuning of CaP cell bodies to ensure proper exit points of motor axons.     

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca²⁺ dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca²⁺ mediated by the plasmalemmal Na⁺/Ca²⁺ exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca²⁺ influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca²⁺ dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.     

Sema3a1 guides spinal motor axons in a cell- and stage-specific manner in zebrafish

In order for axons to reach their proper targets, both spatiotemporal regulation of guidance molecules and stepwise control of growth cone sensitivity to guidance molecules is required. Here, we show that, in zebrafish, Sema3a1, a secreted class 3 semaphorin, plays an essential role in guiding the caudal primary (CaP) motor axon that pioneers the initial region of the motor pathway. The expression pattern of Sema3a1 suggests that it delimits the pioneer CaP axons to the initial, common pathway via a repulsive action, but then CaP axons become insensitive to Sema3a1 beyond the common pathway. Indeed, nrp1a, which probably encodes a component of the Sema3a1 receptor, is specifically expressed by CaP during the early part of its outgrowth but not during later stages when extending into sema3a1-expressing muscle cells. To examine this hypothesis directly, expression of sema3a1 and/or nrp1a was manipulated in several ways. First, antisense knockdown of Sema3a1 induced CaP axons to branch excessively, stall and/or follow aberrant pathways. Furthermore, dynamic analysis showed they extended more lateral filopodia and often failed to pause at the horizontal myoseptal choice point. Second, antisense knockdown of Nrp1a and double knockdown of Nrp1a/Sema3a1 induced similar outgrowth defects in CaP. Third, CaP axons were inhibited by focally misexpressed sema3a1 along the initial common pathway but not along their pathway beyond the common pathway. Thus, as predicted, Sema3a1 is repulsive to CaP axons in the common region of the pathway, but not beyond the common pathway. Fourth, induced ubiquitous overexpression of sema3a1 caused the CaP axons but not the other primary motor axons to follow aberrant pathways. These results suggest that the repulsive response to Sema3a1 of the primary motor axons along the common pathway is both cell-type specific and dynamically regulated, perhaps via regulation of nrp1a.     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.     

Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.     

Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2)

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.     

CRMP2 Protein SUMOylation Modulates NaV1.7 Channel Trafficking

Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.     

CRMP2 is necessary for Neurofibromatosis type 1 related pain

Neurofibromatosis type 1 (NF1) is one of the most common genetic diseases, affecting roughly 1 in 3000 individuals. As a multisystem disorder, it affects cognitive development, as well as bone, nerve and muscle constitution. Peripheral neuropathy in NF1 constitutes a potentially severe clinical complication and is associated with increased morbidity and mortality. The discovery of effective therapies for Neurofibromatosis type 1 (NF1) pain depends on mechanistic understanding that has been limited, in part, by the relative lack of availability of animal models relevant to NF1 pain. We have used intrathecal targeted editing of Nf1 in rats to provide direct evidence of a causal relationship between neurofibromin and pain responses. We demonstrated that editing of neurofibromin results in functional remodeling of peripheral nociceptors characterized by enhancement of interactions of the tetrodotoxin-sensitive (TTX-S) Na⁺ voltage-gated sodium channel (NaV1.7) and the collapsin response mediator protein 2 (CRMP2). Collectively, these peripheral adaptations increase sensory neuron excitability and release of excitatory transmitters to the spinal dorsal horn to establish and maintain a state of central sensitization reflected by hyperalgesia to mechanical stimulation of the hindpaw. The data presented here shows that CRMP2 inhibition is sufficient to reverse the dysregulations of voltage-gated ion channels and neurotransmitter release observed after Nf1 gene editing. The concordance in normalization of ion channel dysregulation by a CRMP2-directed strategy and of hyperalgesia supports the translational targeting of CRMP2 to curb NF1-related pain.     

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy,Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.     

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch‐evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA‐type glutamate receptor activation. Transection experiments suggest that the circuits required for touch‐evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA‐type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Functional analysis of zebrafish GDNF

We have identified zebrafish orthologues of glial cell line-derived neurotrophic factor (GDNF) and the ligand-binding component of its receptor GFRalpha1. We examined the mRNA expression pattern of these genes in the developing spinal cord primary motor neurons (PMN), kidney, and enteric nervous systems (ENS) and have identified areas of correlated expression of the ligand and the receptor that suggest functional significance. Many aspects of zebrafish GDNF expression appear conserved with those reported in mouse, rat, and avian systems. In the zebrafish PMN, GFRalpha1 is only expressed in the CaP motor neuron while GDNF is expressed in the ventral somitic muscle that it innervates. To test the functional significance of this correlated expression pattern, we ectopically overexpressed GDNF in somitic muscle during the period of motor axon outgrowth and found specific perturbations in the pattern of CaP axon growth. We also depleted GDNF protein in zebrafish embryos using morpholino antisense oligos and found that GDNF protein is critical for the development of the zebrafish ENS but appears dispensable for the development of the kidney and PMN.     

NEFLb impairs early nervous system development via regulation of neuron apoptosis in zebrafish

Neurofilament light chain (NEFL), a subunit of neurofilament, has been shown to play an important role in pathogenic neurodegenerative disease and in radial axonal growth. However, information remains largely lacking regarding the function of NEFL in early development to date. In this study, we demonstrated the presence of two nefl genes, nefla and neflb, in zebrafish, generated by fish-specific third round genome duplication. These duplicated nefl genes were predominantly expressed in the nervous system with an overlapping and distinct expression pattern. Both gene knockdown and rescue experiments show that it was neflb rather than nefla that played an indispensable role in nervous system development. It was also found that neflb knockdown resulted in striking apoptosis of the neurons in the brain and spinal cord, leading to morphological defects such as brain structure disorder and trunk bending. Thus, we report a previously uncharacterized role of NEFL that NEFLb impairs the early development of zebrafish nervous system via regulation of the neuron apoptosis in the brain and spinal cord.