Ectodomain shedding of the neural recognition molecule CHL1 by the metalloprotease-disintegrin ADAM8 promotes neurite outgrowth and suppresses neuronal cell death

The neural cell adhesion molecule "close homologue of L1," termed CHL1, has functional importance in the nervous system. CHL1 is expressed as a transmembrane protein of 185 kDa, and ectodomain shedding releases soluble fragments relevant for its physiological function. Here we describe that ADAM8, a member of the family of metalloprotease disintegrins cleaves a CHL1-Fc fusion protein in vitro at two sites corresponding to release of the extracellular domain of CHL1 in fibronectin (FN) domains II (125 kDa) and V (165 kDa), inhibited by batimastat (BB-94). Cleavage of CHL1-Fc in the 125-kDa fragment was not detectable under non-reducing conditions arguing that cleavage resulting in the 165-kDa fragment is more relevant in releasing soluble CHL1 in vivo. In cells transfected with full-length ADAM8, membrane proximal cleavage of CHL1 was similar and not stimulated by phorbol ester 12-O-tetradecanoylphorbol-13-acetate and pervanadate. No cleavage of CHL1 was observed in cells expressing either inactive ADAM8 with a Glu330 to Gln exchange (EQ-A8), or active ADAM10 and ADAM17. Consequently, processing of CHL1 was hardly detectable in brain extracts of ADAM8-deficient mice but enhanced in a neurodegenerative mouse mutant. CHL1 processed by ADAM8 in supernatants of COS-7 cells and in co-culture with cerebellar granule neurons was very potent in stimulating neurite outgrowth and suppressing neuronal cell death, not observed in cells co-transfected with CHL1/EQ-A8, CHL1/ADAM10, or CHL1/ADAM17. Taken together, we propose that ADAM8 plays an important role in physiological and pathological cell interactions by a specific release of functional CHL1 from the cell surface.     

The cell recognition molecule L1 and cognition

Cell adhesion molecules were classically known to be key players in a whole array of biological functions during development through cell-cell and cell-substratum adhesion. Accumulating evidence suggests, however, that the functional significance of adhesion molecules may extend well beyond embryonic development into adult stage. The aim of this article is to review our current knowledge on the implications of adhesion molecules in various cognitive processes. Particular emphasis was placed on the immunoglobulin superfamily of adhesion molecules, which are found to be involved in activity-dependent plasticity of the nervous system, and hence promising candidates in modulating cognitive functions.     

Loss of cell adhesion molecule CHL1 improves homeostatic adaptation and survival in hypoxic stress

Close homologue of L1 (CHL1) is a transmembrane cell adhesion molecule that is critical for brain development and for the maintenance of neural circuits in adults. Recent studies revealed that CHL1 has diverse roles and is involved in the regulation of recovery after spinal cord injury. CHL1 expression was downregulated in the cerebral cortex, hypothalamus, and brain stem after the induction of acute hypoxia (AH). In the current study, we sought to address the role of CHL1 in regulating homeostasis responses to hypoxia using CHL1-knockout (CHL1^−/−) mice. We found that, compared with wild-type littermates, CHL1^−/− mice showed a dramatically lower mortality rate and an augmented ventilatory response after they were subjected to AH. Immunofluorescence staining revealed that CHL1 was expressed in the carotid body (CB), the key oxygen sensor in rodents, and CHL1 expression level in the CB as assayed by western blot was decreased after hypoxic exposure. The number of glomus cells and the expression of tyrosine hydroxylase (a marker for glomus cells) in the CB of CHL1^−/− mice appeared to be increased compared with CHL1^+/+ mice. In addition, in the ex vivo CB preparation, hypoxia induced a significantly greater afferent nerve discharge in CHL1^−/− mice compared with CHL1^+/+ mice. Furthermore, the arterial blood pressure and plasma catecholamine levels of CHL1^−/− mice were also significantly higher than those of CHL1^+/+ mice. Our findings first demonstrate that CHL1 is a novel intrinsic factor that is involved in CB function and in the ventilatory response to AH.     

细胞粘附分子CHL1缺失对炎症性肠病的影响

目的：探讨细胞粘附分子CHL1缺失对炎症性肠病（IBD）的影响。方法：采用葡聚糖硫酸钠（DSS）建立CHL1（-/-）炎症性肠病小鼠模型,将10只C57/BL6为遗传背景的CHL1（+/+）型小鼠分为对照组、DSS模型组（n=5）;将10只C57/BL6为背景的CHL （-/-）型小鼠分为CHL1缺失组、CHL1缺失DSS模型组（n=5）。DSS模型组和CHL1缺失DSS模型组自主饮用7 d含1.5% DSS蒸馏水,随后改用蒸馏水饲喂2 d;对照组和CHL1缺失组连续饮用蒸馏水9 d,观察小鼠体重、血便、生存率及结肠组织长度的变化。结果：CHL1缺失DSS模型组小鼠在炎症性肠病的发生中便血明显,从应激的第7天开始,体重明显减轻,在第9天,CHL1缺失DSS模型组型小鼠死亡率明显增加;结肠长度明显缩短,损伤加重。结论：细胞粘附分子CHL1缺失加重炎症性肠病的发生。     

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.     

Co-cultures of inferior olive and cerebellum: Electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca²⁺-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Spatial and temporal analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites

We have used the calcium indicator dye arsenazo III, together with a photodiode array, to record intracellular calcium changes simultaneously from all regions of individual guinea pig cerebellar Purkinje cells in slices. The optical signals, recorded with millisecond time resolution, are good indicators of calcium-dependent electrical events. For many cells the sensitivity of the recordings was high enough to detect signals from each array element without averaging. Consequently, it was possible to use these signals to follow the complex spatial and temporal patterns of plateau and spike potentials. Calcium entry corresponding to action potentials was detected from all parts of the dendritic field including the fine spiny branchlets, demonstrating that calcium action potentials spread over the entire arbor. Usually, the entire dendritic tree fired at once. But sometimes only restricted areas had signals at any one moment with transients detected in different regions at other times. In one cell, six separate zones were distinguished. These results show that calcium action potentials could be regenerative in some dendrites and could fail to propagate into others. Signals from plateau potentials were also detected from extensive areas in the dendritic field but were always smaller than those caused by a burst of action potentials.     

Co-cultures of inferior olive and cerebellum: electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons.

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca(2+)-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Neurotrophic regulation of retinal ganglion cell synaptic connectivity: from axons and dendrites to synapses

This review highlights important events during the morphological development of retinal ganglion cells (RGCs), focusing on mechanisms that control axon and dendritic arborization as a means to understand synaptic connectivity with special emphasis on the role of neurotrophins during structural and functional development of RGCs. Neurotrophins and their receptors participate in the development of visual connectivity at multiple levels. In the visual system, neurotrophins have been shown to exert various developmental influences, from guiding the morphological differentiation of neurons to controlling the functional plasticity of visual circuits. This review article examines the role of neurotrophins, and in particular of BDNF, during the morphological development of RGCs, and discusses potential interactions between activity and neurotrophins during development of neuronal connectivity.     

Immunocytological characterization of the expression of cell adhesion molecule L1 during early innervation of mouse otocysts

Summary Doubts exist as to whether afferent nerve fibers exert a neurotrophic effect on the differentiation of sensory cells in the developing vestibular neuroepithelium. To determine whether innervation of hair cells precedes their differentiation, we have used the L1 adhesion molecule as a marker for axons. The detection of L1 on afferent axons in the otic vesicle of mouse embryos on gestation day 11 shows that nerve fibers penetrate the neuroepithelium before the sensory cells differentiate. L1-immunoreactivity of nerve endings also reveals the considerable fiber ramification on gestation days 14 and 15, i.e., corresponding to the first stages of sensory cell differentiation. The expression of L1 at successive stages of nerve fiber growth in the neuroepithelium, such as fasciculation and ramification, is not consistent with the previous role proposed for L1 as a fascicule-promoting factor and raises the possibility that other mechanisms are involved in L1 mediaded adhesion.     

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.     

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule

Background  

Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines.     

Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1

The effects of L1-Fc and CHL1-Fc fusion proteins on neuronal survival were investigated. Cerebellar granule neurons of mouse and hippocampal neurons of rat embryo undergo apoptosis when cultured in serum-free medium. Treatment with chimeric proteins containing the extracellular domains of the neural adhesion molecules L1 or CHL1 fused to the Fc region of human immunoglobulin significantly enhanced the survival of neurons. Compared to the control, the percentage of surviving neurons increased about 60% and 45% with L1 and CHL1 fusion proteins, respectively. A fusion protein containing the extracellular domain of NCAM had no effect on survival. The L1 and CHL1 fusion proteins were effective both in soluble form or when offered as a substrate, with the maximal effect at about 1 microg/mL. To explore the intracellular events related to the neuronal survival effects of L1-Fc fusion protein, Bcl-2 and c-Jun expression were analyzed by Western blotting. The level of Bcl-2 in cerebellar granule neurons was increased by treatment with L1-Fc at both 1 and 5 days of culture. The level of c-Jun was not significantly affected at the early time point and was reduced by L1-Fc fusion protein after long-term culture. The results demonstrate that the neural adhesion molecule L1 and its relative CHL1 are potential neuronal survival factors for neurons of the central nervous system. Bcl-2 may serve as one of the intracellular mediators of the neuronal survival effects of L1.     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.