硫酸软骨素蛋白多糖与神经系统的发育和再生

硫酸软骨素蛋白多糖（chondroitin sulfate proteoglycans,CSPGs）是中枢神经系统（CNS）细胞外基质中的重要组成成分,在CNS的发育、成熟后正常功能的维持中发挥重要功能,如发育中影响神经细胞的迁移和轴突生长,成年后参与神经可塑性的控制等;而病理条件下,如CNS受损后又可做为胶质瘢痕的重要组分抑制受损神经的再生。研究发现,用酶降解CSPGs的糖氨多糖链或阻断其合成可以有效地削弱CSPGs对受损神经的抑制作用,促进轴突再生。然而,精确调控CSPGs特定时空表达模式的分子机制,以及功能发挥所涉及的完整信号转导通路还有待进一步研究。     

Control of axonal growth and regeneration of sensory neurons by the p110delta PI 3-kinase

The expression and function of the 8 distinct catalytic isoforms of PI 3-kinase (PI3K) in the nervous system are unknown. Whereas most PI3Ks have a broad tissue distribution, the tyrosine kinase-linked p110delta isoform has previously been shown to be enriched in leukocytes. Here we report that p110delta is also highly expressed in the nervous system. Inactivation of p110delta in mice did not affect gross neuronal development but led to an increased vulnerability of dorsal root ganglia neurons to exhibit growth cone collapse and decreases in axonal extension. Loss of p110delta activity also dampened axonal regeneration following peripheral nerve injury in adult mice and impaired functional recovery of locomotion. p110delta inactivation resulted in reduced neuronal signaling through the Akt protein kinase, and increased activity of the small GTPase RhoA. Pharmacological inhibition of ROCK, a downstream effector of RhoA, restored axonal extension defects in neurons with inactive p110delta, suggesting a key role of RhoA in p110delta signaling in neurons. Our data identify p110delta as an important signaling component for efficient axonal elongation in the developing and regenerating nervous system.     

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.     

Ultramicroscopy Reveals Axonal Transport Impairments in Cortical Motor Neurons at Prion Disease

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.     

Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals.

In adult mammals, the severing of the optic nerve near the eye is followed by a loss of retinal ganglion cells (RGCs) and a failure of axons to regrow into the brain. Experimental manipulations of the non-neuronal environment of injured RGCs enhance neuronal survival and make possible a lengthy axonal regeneration that restores functional connections with the superior colliculus. These effects suggest that injured nerve cells in the mature central nervous system (CNS) are strongly influenced by interactions with components of their immediate environment as well as their targets. Under these conditions, injured CNS neurons can express capacities for growth and differentiation that resemble those of normally developing neurons. An understanding of this regeneration in the context of the cellular and molecular events that influence the interactions of axonal growth cones with their non-neuronal substrates and neuronal targets should help in the further elucidation of the capacities of neuronal systems to recover from injury.     

Semaphorin-neuropilin-1 interactions in plasticity and regeneration of adult neurons

During development, axonal growth cones are guided to their appropriate targets by many attractive and repulsive cues. It has become increasingly clear over the last few years that how the growth cone responds to these cues depends both on the molecular nature of the cue and on the internal state of the neuron. The unexpected result is that the same molecule can act as an attractor or as a repellent. A number of guidance cues used by neurons during development are retained in the adult nervous system, where their function is often still unclear. Most of these molecules are implicated in plasticity in the adult nervous system and can play a role (sometimes maladaptive) in neuronal regeneration after injury. A group of axonal guidance cues that has been well studied in development is the semaphorin family of secreted and membrane-anchored proteins, which has been implicated in axon steering, fasciculation, branching and synapse formation. This review focuses on semaphorin-3A (probably the best-characterized semaphorin) and its receptors (in particular neuropilin-1) in the adult nervous system and argues that semaphorin-3A plays a role in the maintenance and regeneration of adult sensory neurons.     

Neurotrophic factors in combinatorial approaches for spinal cord regeneration

Axonal regeneration is inhibited by a plethora of different mechanisms in the adult central nervous system (CNS). While neurotrophic factors have been shown to stimulate axonal growth in numerous animal models of nervous system injury, a lack of suitable growth substrates, an insufficient activation of neuron-intrinsic regenerative programs, and extracellular inhibitors of regeneration limit the efficacy of neurotrophic factor delivery for anatomical and functional recovery after spinal cord injury. Thus, growth-stimulating factors will likely have to be combined with other treatment approaches to tap into the full potential of growth factor therapy for axonal regeneration. In addition, the temporal and spatial distribution of growth factors have to be tightly controlled to achieve biologically active concentrations, to allow for the chemotropic guidance of axons, and to prevent adverse effects related to the widespread distribution of neurotrophic factors. Here, we will review the rationale for combinatorial treatments in axonal regeneration and summarize some recent progress in promoting axonal regeneration in the injured CNS using such approaches.     

The common properties of neurogenesis in the adult brain: from invertebrates to vertebrates

Until recently, it was believed that adult brains were unable to generate any new neurons. However, it is now commonly known that stem cells remain in the adult central nervous system and that adult vertebrates as well as adult invertebrates are currently adding new neurons in some specialized structures of their central nervous system. In vertebrates, the subventricular zone and the dentate gyrus of the hippocampus are the sites of neuronal precursor proliferation. In some insects, persistent neurogenesis occurs in the mushroom bodies, which are brain structures involved in learning and memory and considered as functional analogues of the hippocampus. In both vertebrates and invertebrates, secondary neurogenesis (including neuroblast proliferation and neuron differentiation) appears to be regulated by hormones, transmitters, growth factors and environmental cues. The functional implications of adult neurogenesis have not yet been clearly demonstrated and comparative study of the various model systems could contribute to better understand this phenomenon. Here, we review and discuss the common characteristics of adult neurogenesis in the various animal models studied so far.     

Role of neurotrophic factors in development

Neurotrophic factors are molecules which promote and regulate neuronal survival in the developing nervous system. They are distinguished from ubiquitous metabolites necessary for cellular maintenance and growth by their specificity: each neurotrophic factor promotes the survival of only certain kinds of neurons during a particular stage of their development. In addition, it has been argued that neurotrophic factors are involved in many other aspects of neuronal development ranging from axonal guidance to regulation of neurotransmitter synthesis. Recent developmental studies and the use of specific molecular probes have greatly clarified the role of these molecules.     

Schwann Cell Exosomes Mediate Neuron–Glia Communication and Enhance Axonal Regeneration

The functional and structural integrity of the nervous system depends on the coordinated action of neurons and glial cells. Phenomena like synaptic activity, conduction of action potentials, and neuronal growth and regeneration, to name a few, are fine tuned by glial cells. Furthermore, the active role of glial cells in the regulation of neuronal functions is underscored by several conditions in which specific mutation affecting the glia results in axonal dysfunction. We have shown that Schwann cells (SCs), the peripheral nervous system glia, supply axons with ribosomes, and since proteins underlie cellular programs or functions, this dependence of axons from glial cells provides a new and unexplored dimension to our understanding of the nervous system. Recent evidence has now established a new modality of intercellular communication through extracellular vesicles. We have already shown that SC-derived extracellular vesicles known as exosomes enhance axonal regeneration, and increase neuronal survival after pro-degenerative stimuli. Therefore, the biology nervous system will have to be reformulated to include that the phenotype of a nerve cell results from the contribution of two nuclei, with enormous significance for the understanding of the nervous system in health and disease.     

Coordinate regulation of the expression of axonal proteins by the axonal microenvironment

The axonal functions that act in the formation of the neuronal network have been shown to occur in close interdependence with the tissue that surrounds the growing axons. However, little is known about the molecular building blocks underlying axonal functions, although more than 400 axonal proteins have been identified. In view of the existence of such a large number of axonal proteins, we have initiated a project to determine the molecules involved in the implementation of particular axonal functions by a selective approach. On the assumption that plasticity in the expression of axonal functions in response to specific features of the local axonal environment may be based on changes in the expression of particular axonal proteins, the axonal proteins of dorsal root ganglion (DRG) neurons were screened for those whose expression responds to environmental influences. DRG neurons were grown in a compartmental cell system that offers separate access to neuronal somas and to their axons and the axons were locally exposed to different populations of cells from the peripheral or central nervous system. The axonal proteins were metabolically labeled and subjected to two-dimensional gel electrophoresis. Computerized quantitation of the individual axonal proteins revealed that the cocultured cells modulate the synthesis of a few axonal proteins of DRG neurons differentially. The data on the abundance of the newly expressed proteins under varying local environmental conditions were condensed as expression profiles. Comparison of expression profiles and cluster analysis of quantitative gel analysis data revealed that the environmentally modulated proteins subdivide into clusters with common distinct expression profiles under the influence of nonneuronal cells from the peripheral nervous system, nonneuronal cells of the central nervous system, and spinal cord cells, which are composed of neurons and nonneuronal cells. By means of this new, characteristic attribute assigned to environmentally modulated axonal proteins, working hypotheses were made as to their functional role.     

Mitochondrial Regulation of Neuronal Plasticity

The structure and function of neurons is dynamic during development and in adaptive responses of the adult nervous system to environmental demands. The mechanisms that regulate neuronal plasticity are poorly understood, but are believed to involve neurotransmitter and neurotrophic factor signaling pathways. In the present article, I review emerging evidence that mitochondria play important roles in regulating developmental and adult neuroplasticity. In neurons, mitochondria are located in axons, dendrites, growth cones and pre- and post-synaptic terminals where their movements and functions are regulated by local signals such as neurotrophic factors and calcium influx. Mitochondria play important roles in fundamental developmental processes including the establishment of axonal polarity and the regulation of neurite outgrowth, and are also involved in synaptic plasticity in the mature nervous system. Abnormalities in mitochondria are associated with neurodegenerative and psychiatric disorders, suggesting a therapeutic potential for approaches that target mitochondrial mechanisms. Special issue dedicated to John P. Blass.     

Sculpting the nervous system: glial control of neuronal development

Glial cells are not passive spectators during nervous system assembly, rather they are active participants that exert significant control over neuronal development. Well-established roles for glia in shaping the developing nervous system include providing trophic support to neurons, modulating axon pathfinding, and driving nerve fasciculation. Exciting recent studies have revealed additional ways in which glial cells also modulate neurodevelopment. Glial cells regulate the number of neurons at early developmental stages by dynamically influencing neural precursor divisions, and at later stages by promoting neuronal cell death through engulfment. Glia also participate in the fine sculpting of neuronal connections by pruning excess axonal projections, shaping dendritic spines, and secreting multiple factors that promote synapse formation and functional maturation. These recent insights provide further compelling evidence that glial cells, through their diverse cellular actions, are essential contributors to the construction of a functionally mature nervous system.     

The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function.     

Insulin-like peptides are not involved in maturation or functional recovery of neural circuits in the locust flight system

We sought to manipulate maturation and functional recovery of locust flight circuitry by treating locusts with pharmacological doses of bovine anti-insulin and insulin. Anti-insulin treatment of maturing locusts caused reduced growth of the thoracic nervous system, lower body weight, and softer cuticles compared with control locusts. We were unable to block either maturation or recovery of flight circuitry with anti-insulin. We propose that insulin-related peptides are involved in growth and cuticular changes during adult maturation, but have no role in promoting neuronal sprouting during this period or as a result of injury.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Myosin function in nervous and sensory systems

Development of the nervous system requires remarkable changes in cell structure that are dependent upon the cytoskeleton. The importance of specific components of the neuronal cytoskeleton, such as microtubules and neurofilaments, to neuronal function and development has been well established. Recently, increasing focus has been put on understanding the functional role of the actin cytoskeleton in neurons. Important modulators of the actin cytoskeleton are the large family of myosins, many of which (classes I, II, III, V, VI, VII, IX, and XV; Fig. 1) are expressed in developing neurons or sensory cells. Myosins are force-producing proteins that have been implicated in a wide variety of cellular functions in the developing nervous system, including neuronal migration, process outgrowth, and growth cone motility, as well as other aspects of morphogenesis, axonal transport, and synaptic and sensory functions. We review the roles that neuronal myosins play in these functions with particular focus on the first three events listed above, as well as sensory function.     

The evolving roles of axonally synthesized proteins in regeneration

Work emerging during the past decade has shown that axons, similar to dendrites, are capable of autonomously generating new proteins through translation of localized mRNAs. Even in mammals, neurons maintain the ability to target mRNAs and translational machinery into the axonal compartment well into adulthood. The biological functions of axonal protein synthesis in adult neurons are just now being revealed, and recent studies indicate that locally synthesized proteins facilitate regeneration. Local translation, in addition to protein degradation, is needed for growth cone formation after axotomy, for generating a retrogradely transported injury signal, and then to help structurally maintain the growing axon. Regulation of axonal protein synthesis by exogenous stimuli might provide a means to facilitate regeneration for neuronal populations that normally show poor regenerative capacity in the adult nervous system.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.Key Words: motor cortex, neuronal transplantation, embryonic cells, GFP, GFAP, PLP     

Prominent expression of acidic fibroblast growth factor in motor and sensory neurons.

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.