Wnt5a induces Ryk-dependent and -independent effects on callosal axon and dendrite growth

Abstract

The non-canonical Wnt receptor, Ryk, promotes chemorepulsive axon guidance in the developing mouse brain and spinal cord in response to Wnt5a. Ryk has also been identified as a major suppressor of axonal regrowth after spinal cord injury. Thus, a comprehensive understanding of how growing axons and dendrites respond to Wnt5a-mediated Ryk activation is required if we are to overcome this detrimental activity. Here we undertook a detailed analysis of the effect of Wnt5a/Ryk interactions on axonal and dendritic growth in dissociated embryonic mouse cortical neuron cultures, focusing on callosal neurons known to be responsive to Ryk-induced chemorepulsion. We show that Ryk inhibits axonal growth in response to Wnt5a. We also show that Wnt5a inhibits dendrite growth independently of Ryk. However, this inhibition is relieved when Ryk is present. Therefore, Wnt5a-mediated Ryk activation triggers divergent responses in callosal axons and dendrites in the in vitro context.     

Corticospinal tract fibers cross the ephrin-B3-negative part of the midline of the spinal cord after brain injury

The fibers of corticospinal tract (CST), which control fine motor function, predominantly project to the contralateral spinal cord, not recross to the ipsilateral side. Ephrin-B3, which is expressed in the midline of the spinal cord, and its receptor, EphA4, are crucial for preventing CST fibers from recrossing the midline in the developing spinal cord. However, these fibers can cross the midline to the denervated side after a unilateral CST or cortical injury. We determined the reason CST fibers can cross the midline after a cortical injury and the changes in ephrin-B3-EphA4 signaling associated with such a crossing. We first examined axonal sprouting from CST fibers after unilateral ablation of the motor cortex in postnatal and adult mice. CST fibers crossed the midline of the spinal cord after cortical ablation, especially when conducted during the early postnatal period. These fibers were well associated with functional recovery after the injury. We next assessed the mRNA expression of ephrin-B3 and EphA4 before and after the ablation. Surprisingly, no changes were detected in the expression patterns. We found, however, that ephrin-B3 expression in the ventral part of the midline disappeared after postnatal day 9 (P9), but was pronounced along the entire midline before P6. Most of the CST fibers crossed the midline through the ventral region, where ephrin-B3 expression was absent. Our results suggest that ephrin-B3 is not expressed along the entire midline of the spinal cord, and sprouting axons can cross the midline at ephrin-B3-negative areas.     

On the development of the pyramidal tract in the rat

Summary An anterograde tracer study has been made of the developing corticospinal tract (CST) in the rat using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Analysis of normal Rager stained material revealed that corticospinal axons reach upper cervical spinal cord levels at the day of birth (PO). Postnatal rats ranging in age from one (P1) to fourteen (P14) days received multiple WGA-HRP injections into the cortex of their left hemisphere and were allowed to survive for 24 h. The first labeled CST fibers caudally extend into the third thoracic spinal cord segment at P1; into the eighth thoracic segment at P3; into the first or second lumbar segment at P7 and into the second to third sacral segment at Pg. Thus the outgrowth of the leading pioneer fibers of the CST is completed at P9 but later developing axons are continuously added even beyond P9. Quantitative analysis of the amount of label along the length of the outgrowing CST revealed a characteristic pattern of labeling varying with age. The most striking features of that pattern are: (1) the formation of two standing peaks at the level of the cervical and lumbar enlargements respectively and (2) the transient presence of a smaller running peak which moves caudally with the front of the outgrowing bundle. The standing peaks are ascribed to the branching of the axon terminals at both intumescences, whereas the running peak probably arises by the accumulation of tracer within the growth cones at the tips of the outgrowing CST axons. Factors such as the number of axons, the varying axon diameters, the branching collaterals, the presence of varicosities, the transport rate of the tracer, the uptake of the tracer at the injection site, which possibly may affect the amount of label present in both the entire bundle and in the individual axons are discussed. Current research is focused upon an analysis of the relation between the site of injection within the cortex and the pattern of labeling of the CST. A delay of two days was found between the arrival of the CST axons at a particular spinal cord level and their outgrowth into the adjacent spinal gray. However, combined HRP and electronmicroscopic experiments are necessary to determine the factors behind the maturation of the CST as well as the maturation of the spinal gray.     

孕酮对脊髓损伤后神经纤维的保护作用

据报导孕酮可以加强髓鞘蛋白的表达量,稳定神经系统的白质。脊髓内移植背根节神经细胞后,其长出的神经纤维可长距离的在溃变神经纤维的支架上生长。为了研究脊髓损伤后孕酮对受损白质的溃变和再生神经纤维生长的影响,将C57小鼠脊髓胸8(Th8)节段皮质脊髓束横断后,将孕酮溶于芝麻油(10mg/ml)后皮下注射,隔日注射1次,持续2周采用丽春红2R-亮绿染色法显示皮质脊髓束在脊髓内的位置、形态。结果显示:皮下注射孕酮之后皮质脊髓束远端轴突更加完整,排列更加规则、致密,对照组中远端轴突碎裂严重,排列杂乱,纤维丢失。上述结果提示孕酮能够保护小鼠受损的皮质脊髓束远端轴突。     

Molecular mechanisms of axon guidance in the developing corticospinal tract

The great repertoire of movements in higher order mammals comes courtesy of the corticospinal tract (CST) which is able to initiate precise movement of the entire musculature of the axial and limb muscle groups. It forms the longest axonal trajectory in the mammalian central nervous system and its axons must navigate the entire length of the central nervous system--from its origins in the deeper layers of the cerebral cortex down through the cerebral peduncles and brainstem and along the entire length of the spinal cord. This period of navigation is incredibly complex, and relies upon the coordinated regulation of a collection of molecular guidance cues - coming from all of the known major families of guidance cues - the ephrins, slits, Netrins and Semaphorins - that work together to steer the growing axonal tips through the brain and spinal cord. As such a long tract, the CST forms an excellent experimental model to investigate the nature of molecular cues that sequentially guide axons through the central nervous system. Using the rodent as a model system, this review discusses each step of axonal guidance through the major brain regions--starting from the decision to grow ventrally out of the cortical plate to the eventual activity-dependent refinement of circuitry in the spinal grey matter. In recent years, the identification of these guidance cues and their proposed mode of action is beginning to give us a picture at a molecular level of how the CST is guided so accurately over such a long distance.     

Collateralization of the cervical corticospinal tract in the rat

Anterograde staining with Phaseolus vulgaris leucoagglutinin (PHA-L) revealed the spinal arborization pattern of corticospinal tract (CST) fibers in the cervical enlargement of the rat. Within the confines of the pyramidal tract local nets of small fibers are present in addition to the rather large CST fibers with varicosities. CST termination is primarily located in lamina IV and extends into lamina V and VI. Extensive collateralization of CST axons was found interconnecting neurons located both in different horizontal laminae and in subsequent spinal cord segments. This complex pattern of CST collateralization is suggested to add a coordinative role in motor control to this tract both through serial axo-dendritic contacts in the spinal gray and through axo-axonal contacts in the white as well as the gray matter.     

The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

In contrast to peripheral nerves, central axons do not regenerate. Partial injuries to the spinal cord, however, are followed by functional recovery. We investigated the anatomical basis of this recovery and found that after incomplete spinal cord injury in rats, transected hindlimb corticospinal tract (CST) axons sprouted into the cervical gray matter to contact short and long propriospinal neurons (PSNs). Over 12 weeks, contacts with long PSNs that bridged the lesion were maintained, whereas contacts with short PSNs that did not bridge the lesion were lost. In turn, long PSNs arborize on lumbar motor neurons, creating a new intraspinal circuit relaying cortical input to its original spinal targets. We confirmed the functionality of this circuit by electrophysiological and behavioral testing before and after CST re-lesion. Retrograde transynaptic tracing confirmed its integrity, and revealed changes of cortical representation. Hence, after incomplete spinal cord injury, spontaneous extensive remodeling occurs, based on axonal sprout formation and removal. Such remodeling may be crucial for rehabilitation in humans.     

皮质脊髓束的发育和再生

皮质脊髓束(corticosp inal tract,CST)是脊髓中最重要的下行运动传导束,它的受损与临床上的中枢性硬瘫密切相关。CST神经元产生于大脑皮质ⅴ层,在发育过程中有轴突生长过剩及消除现象。不同物种之间CST纤维束生长的时间也不同。GAP-43、CNTF、GDNF等因子在CST的发育过程中起重要作用。CST损伤后,周围的微环境存在抑制性因素,导致其再生困难。目前的再生修复研究大多集中于神经或细胞移植、神经生长因子及抗抑制因子抗体的应用。     

#br# 鸡源Wnt3a融合蛋白表达载体的构建及其对鸡胚脊髓神经前体细胞增殖和轴突形成的影响

目的 构建鸡源Wnt3a 融合蛋白真核表达载体 (pCAG-MCs-Wnt3a-EGFP),并探讨其在鸡胚脊髓发育过程中超表达后对神经前体细胞增殖及轴突形成的影响。 方法 利用分子生物学手段,提取鸡胚脊髓总RNA并获得Wnt3a片段,将其克隆到pCAG-MCs-EGFP载体中构建pCAG-MCs-Wnt3a-EGFP表达载体。在鸡胚发育至2.5～3d (E2.5～E3) 时,利用鸡胚活体电转技术将pCAG-MCs-Wnt3a-EGFP（实验组）和pCAG-MCs-EGFP（对照组）质粒分别转入鸡胚脊髓,E4时取材切片,每组5个胚胎组织,采用免疫荧光染色技术检测Wnt3a和增殖细胞核抗原（PCNA）蛋白表达变化分析Wnt3a与细胞增殖间的关系,根据载体自发绿色荧光蛋白（GFP）观察脊髓神经前体细胞轴突生成情况。 结果 pCAG-MCs-Wnt3a-EGFP表达载体基因测序结果与Gene bank中基因序列一致,将pCAG-MCs-Wnt3a-EGFP导入鸡胚脊髓中发现绿色荧光。在脊髓组织切片水平上,免疫荧光染色结果表明,Wnt3a在鸡胚脊髓中能够超表达。Wnt3a超表达后,与对照组比较,含有轴突的神经元数量明显减少(n=3, P<0.01),而PCNA 的表达量显著增加(n=3, P<0.01)。 结论 成功构建了鸡源性Wnt3a 融合蛋白真核表达载体,并证实Wnt3a在鸡胚发育过程中促进神经前体细胞的增殖并抑制轴突的形成。     

Ipsilateral, ventral corticospinal tract of the adult rat: ultrastructure, myelination and synaptic connections

The corticospinal tract (CST) of the rat is a widely used model system in developmental, physiological, and regeneration studies. The CST of the rat consists of a main tract, that runs in the dorsomedial funiculus and several minor components. We have shown earlier that one of the minor components, the ipsilateral, ventral CST, projects all the way down the spinal cord in the adult rat and single fibers form large terminal arbors in their spinal target areas. Here we investigated its ultrastructure and compared it to that of CST fibers of the main tract. By the use of anterograde axonal tracing with biotin dextran-amine (BDA) and pre-embedding avidin-peroxidase histochemistry we investigated axon diameters and myelination using electron microscopy. Ipsilateral, ventral CST fibers were found to run in the ventral funiculus close to the midline. They were intermingled with heavily myelinated large diameter axons, presumably reticulospinal, vestibulospinal, or tectospinal fibers. Ipsilateral, ventral CST fibers were of small diameter (0.68 m, ±0.04) and about [frac34] of them were moderately myelinated (9.64 ± 0.7 layers of myelin). Co-localization of a rhodamine-dextrane anterograde tracer with the presynaptic marker synaptophysin using confocal microscopy and electron microscopy revealed varicosities on terminal arborisations to be presynaptic boutons and clearly demonstrated contacts to neurons in intermediate laminae of the spinal cord at lumbar spinal levels. This study extends our earlier work indicating that the ipsilateral, ventral CST component of the adult rat is a morphologically complete CST component and may perform similar functions to the main CST component.     

Deficiency of the Wnt receptor Ryk causes multiple cardiac and outflow tract defects

Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk^?/? embryos. Ryk^?/? mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.     

The effect of systemic PTEN antagonist peptides on axon growth and functional recovery after spinal cord injury

Knockout studies suggest that PTEN limits the regenerative capacities of CNS axons as a dominant antagonist of PI3 kinase, but the transgenic approach is not feasible for treating patients. Although application of bisperoxovanadium may block PTEN function, it is a general inhibitor of phosphotyrosine phosphatases and may target enzymes other than PTEN, causing side effects and preventing firm conclusions about PTEN inhibition on regulating neuronal growth. A pharmacological method to selectively suppress PTEN post-injury could be a valuable strategy for promoting CNS axon regeneration. We identified PTEN antagonist peptides (PAPs) by targeting PTEN critical functional domains and evaluated their efficacy for promoting axon growth. Four PAPs (PAP 1–4) bound to PTEN protein expressed in COS7 cells and blocked PTEN signaling in vivo. Subcutaneous administration of PAPs initiated two days after dorsal over-hemisection injury significantly stimulated growth of descending serotonergic fibers in the caudal spinal cord of adult mice. Systemic PAPs induce significant sprouting of corticospinal fibers in the rostral spinal cord and limited growth of corticospinal axons in the caudal spinal cord. More importantly, PAP treatment enhanced recovery of locomotor function in adult rodents with spinal cord injury. This study may facilitate development of effective therapeutic agents for CNS injuries.     

大鼠自体嗅成鞘细胞移植治疗脊髓损伤的实验研究

目的探讨自体外周嗅成鞘细胞对脊髓损伤修复的可能作用和促进受损轴突再生的能力,为临床应用自体OECs修复脊髓损伤进行可行性研究.方法以改良的Allen法建立脊髓（T9～10节段）损伤模型后,将动物分为自体OECs悬液组和Hanks液对照组,分别移植含自体OECs的细胞悬液和Hanks液,以神经丝蛋白（NF）和神经生长因子受体蛋白（NGFR p75）免疫荧光双标染色,激光共聚焦显微镜检测移植效果;通过BBB行为学评分记录两组动物后肢恢复功能.结果镜下观察可见自体OECs悬液组动物脊髓损伤处的空洞较对照组明显缩小.而自体OECs悬液组免疫荧光标记纤维中夹有NGFRp75标记的OECs,且随标记的NF纤维走行方向排列.行为学观察可见自体OECs悬液组的大鼠后肢运动功能较对照组有显著恢复.结论自体OECs悬液多点注射有助于脊髓损伤部位神经纤维的再生.     

Functional changes in genetically dysmyelinated spinal cord axons of shiverer mice: role of juxtaparanodal Kv1 family K+ channels

Axonal dysfunction after spinal cord injury (SCI) and other types of neurotrauma is associated with demyelination and exposure of juxtaparanodal K+ channels. In this study, sucrose gap electrophysiology using selective and nonselective K+ channel blockers, confocal immunohistochemistry, and Western blotting were used to study the role of Kv1.1 and Kv1.2 K+ channel subunits in dysmyelination-induced spinal cord axonal dysfunction in shiverer mice, which lack the gene encoding myelin basic protein (MBP) and exhibit incomplete myelin sheath formation on CNS axons. The shiverer spinal cord axons exhibited smaller amplitude of compound action potentials (CAPs), reduced conduction velocity, reduced excitability, and greater degree of high-frequency conduction failure. The "fast" K+ channel blocker 4-aminopyridine, the toxin DTX-I, which targets the Kv1.1 and Kv1.2, but not DTX- K, which has higher selectivity for Kv1.1, increased the amplitude and area of CAPs of shiverer mice spinal cord axons but had insignificant effects in wild-type mice. Confocal immunohistochemistry showed that, unlike wild-type mice, which have a precise juxtaparanodal localization of the Kv1.l and Kv1.2 K+ channel subunits, shiverer mouse axons displayed a dispersed distribution of these subunits along the internodes. In contrast, the Kv1.l and Kv1.2 subunits, Na+ channels remained highly localized to the nodal regions. Western blotting showed an increased expression of Kv 1.1 and 1.2 in the shiverer mouse spinal cord. These results provide evidence that the neurological deficits associated with myelin deficiency reflect the altered distribution and expression of the K+ channel subunits Kv1.l and Kv1.2 along the internodes of spinal cord axons associated with the biophysical consequences caused by alterations in the myelin sheaths.     

Probable corticospinal tract control of spinal cord plasticity in the rat

Descending activity from the brain shapes spinal cord reflex function throughout life, yet the mechanisms responsible for this spinal cord plasticity are poorly understood. Operant conditioning of the H-reflex, the electrical analogue of the spinal stretch reflex, is a simple model for investigating these mechanisms. An earlier study in the Sprague-Dawley rat showed that acquisition of an operantly conditioned decrease in the soleus H-reflex is not prevented by mid-thoracic transection of the ipsilateral lateral column (LC), which contains the rubrospinal, reticulospinal, and vestibulospinal tracts, and is prevented by transection of the dorsal column, which contains the main corticospinal tract (CST) and the dorsal column ascending tract (DA). The present study explored the effects of CST or DA transection on acquisition of an H-reflex decrease, and the effects of LC, CST, or DA transection on maintenance of an established decrease. CST transection prior to conditioning prevented acquisition of H-reflex decrease, while DA transection did not do so. CST transection after H-reflex decrease had been acquired led to gradual loss of the decrease over 10 days, and resulted in an H-reflex that was significantly larger than the original, naive H-reflex. In contrast, LC or DA transection after H-reflex decrease had been acquired did not affect maintenance of the decrease. These results, in combination with the earlier study, strongly imply that in the rat the corticospinal tract (CST) is essential for acquisition and maintenance of operantly conditioned decrease in the H-reflex and that other major spinal cord pathways are not essential. This previously unrecognized aspect of CST function gives insight into the processes underlying acquisition and maintenance of motor skills and could lead to novel methods for inducing, guiding, and assessing recovery of function after spinal cord injury.     

Conditioned H-reflex increase persists after transection of the main corticospinal tract in rats

The brain shapes spinal cord function throughout life. Operant conditioning of the H-reflex, the electrical analog of the spinal stretch reflex (SSR), is a relatively simple model for exploring the spinal cord plasticity underlying this functional change and may provide a new method for modifying spinal cord reflexes after spinal cord injury. In response to an operant conditioning protocol, rats can gradually increase (i.e., up-training mode) or decrease (i.e., down-training mode) the soleus H-reflex. This study explored the effects of midthoracic transection of the ipsilateral lateral column (LC) (rubrospinal, vestibulospinal, and reticulospinal tracts), the dorsal column corticospinal tract (CST), or the dorsal column ascending tract (DA) on maintenance of an H-reflex increase that has already occurred. Rats were implanted with EMG electrodes in the right soleus muscle and a nerve-stimulating cuff on the right posterior tibial nerve. After initial (i.e., control) H-reflex size was determined, the rats were exposed for 50 days to the up-training mode, in which reward was given when the H-reflex was above a criterion value. H-reflex size gradually rose to 168 +/- 12% (mean +/- SE) of its initial value. Each rat then received an LC, CST, or DA transection and continued under the up-training mode for 50 more days. None of the transections abolished the H-reflex increase. H-reflex size increased further to 197 +/- 19% of its initial value and did not differ significantly among LC, CST, and DA rats (P > 0.78 by ANOVA). Although earlier studies show that the main CST is needed for acquisition of H-reflex up-training and down-training and for maintenance of down-training, this study shows that it is not needed for maintenance of up-training. It adds to the evidence that H-reflex conditioning changes the spinal cord and that the spinal cord plasticity associated with up-training is different from that associated with down-training.     

双色荧光示踪鸡胚脊髓两侧连合纤维投射研究方法的建立

目的建立1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的实验方法。方法鸡胚孵育至胚龄2.5~3d,通过鸡胚活体原位电转基因技术将携带有报告基因绿色荧光蛋白(GFP)的质粒(p CAGGS-GFP)准确注射到鸡胚脊髓腔,实现定时、定位活体电转基因。转染后继续孵育至6d,取GFP阳性表达的胚胎,部分做脊髓横向切片,部分利用open-book技术将脊髓展开观察连合纤维的发育情况,每组至少取3个标本。其后在脊髓非转染侧连合神经元所在之处,点状注射Di I乙醇溶液,封片后于4℃避光孵育3d,在荧光显微镜下观察脊髓连合纤维投射情况。结果脊髓横切及open-book结果显示,鸡胚脊髓GFP阳性转染侧的神经元轴突穿过底板投射到脊髓对侧;同时在open-book结果中还可观察到,转染侧轴突穿过底板后分别沿腹索和外侧索向头尾部投射;Di I标记的非转染侧连合神经元轴突也同样穿过底板投射到对侧,并在侧索白质内延伸。结论本实验成功建立了1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的研究方法,为研究脊髓神经发育提供技术保障。