Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway

The failure of axons to regenerate is a major obstacle for functional recovery after central nervous system (CNS) injury. Removing extracellular inhibitory molecules results in limited axon regeneration in vivo. To test for the role of intrinsic impediments to axon regrowth, we analyzed cell growth control genes using a virus-assisted in vivo conditional knockout approach. Deletion of PTEN (phosphatase and tensin homolog), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, in adult retinal ganglion cells (RGCs) promotes robust axon regeneration after optic nerve injury. In wild-type adult mice, the mTOR activity was suppressed and new protein synthesis was impaired in axotomized RGCs, which may contribute to the regeneration failure. Reactivating this pathway by conditional knockout of tuberous sclerosis complex 1, another negative regulator of the mTOR pathway, also leads to axon regeneration. Thus, our results suggest the manipulation of intrinsic growth control pathways as a therapeutic approach to promote axon regeneration after CNS injury.     

Cyclic AMP-induced repair of zebrafish spinal circuits

Neurons in the human central nervous system (CNS) are unable to regenerate, as a result of both an inhibitory environment and their inherent inability to regrow. In contrast, the CNS environment in fish is permissive for growth, yet some neurons still cannot regenerate. Fish thus offer an opportunity to study molecules that might surmount the intrinsic limitations they share with mammals, without the complication of an inhibitory environment. We show by in vivo imaging in zebrafish that post-injury application of cyclic adenosine monophosphate can transform severed CNS neurons into ones that regenerate and restore function, thus overcoming intrinsic limitations to regeneration in a vertebrate.     

Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity

The primary circadian pacemaker, in the suprachiasmatic nucleus (SCN) of the mammalian brain, is photoentrained by light signals from the eyes through the retinohypothalamic tract. Retinal rod and cone cells are not required for photoentrainment. Recent evidence suggests that the entraining photoreceptors are retinal ganglion cells (RGCs) that project to the SCN. The visual pigment for this photoreceptor may be melanopsin, an opsin-like protein whose coding messenger RNA is found in a subset of mammalian RGCs. By cloning rat melanopsin and generating specific antibodies, we show that melanopsin is present in cell bodies, dendrites, and proximal axonal segments of a subset of rat RGCs. In mice heterozygous for tau-lacZ targeted to the melanopsin gene locus, beta-galactosidase-positive RGC axons projected to the SCN and other brain nuclei involved in circadian photoentrainment or the pupillary light reflex. Rat RGCs that exhibited intrinsic photosensitivity invariably expressed melanopsin. Hence, melanopsin is most likely the visual pigment of phototransducing RGCs that set the circadian clock and initiate other non-image-forming visual functions.     

EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.     

Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice

In the mammalian retina, a small subset of retinal ganglion cells (RGCs) are intrinsically photosensitive, express the opsin-like protein melanopsin, and project to brain nuclei involved in non-image-forming visual functions such as pupillary light reflex and circadian photoentrainment. We report that in mice with the melanopsin gene ablated, RGCs retrograde-labeled from the suprachiasmatic nuclei were no longer intrinsically photosensitive, although their number, morphology, and projections were unchanged. These animals showed a pupillary light reflex indistinguishable from that of the wild type at low irradiances, but at high irradiances the reflex was incomplete, a pattern that suggests that the melanopsin-associated system and the classical rod/cone system are complementary in function.     

Changing face of microglia

Microglia are resident brain cells that sense pathological tissue alterations. They can develop into brain macrophages and perform immunological functions. However, expression of immune proteins by microglia is not synonymous with inflammation, because these molecules can have central nervous system (CNS)-specific roles. Through their involvement in pain mechanisms, microglia also respond to external threats. Experimental studies support the idea that microglia have a role in the maintenance of synaptic integrity. Analogous to electricians, they are capable of removing defunct axon terminals, thereby helping neuronal connections to stay intact. Microglia in healthy CNS tissue do not qualify as macrophages, and their specific functions are beginning to be explored.     

Seeing more clearly: recent advances in understanding retinal circuitry

Among 10 breakthroughs that Science announced at the end of 2002 was the discovery of a photosensing (melanopsin-containing) retinal ganglion cell (RGC) and its role in entraining the circadian clock. This breakthrough exemplifies the ultimate goal of neuroscience: to understand the nervous system from molecules to behavior. Light-sensing RGCs constitute one of a dozen discrete RGC populations coding various aspects of visual scenes by virtue of their unique morphology, physiology, and coverage of the retina. Interestingly, the function of the melanopsin-containing RGCs in entraining the circadian clock need not involve much retinal processing, making it the simplest form of processing in the retina. This review focuses on recent advances in our understanding of retinal circuitry, visual processing, and retinal development demonstrated by innovative experimental techniques. It also discusses the advantages of using the retina as a model system to address some of the key questions in neuroscience.     

Glial membranes at the node of Ranvier prevent neurite outgrowth

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.     

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.     

GDF11 controls the timing of progenitor cell competence in developing retina

The orderly generation of cell types in the developing retina is thought to be regulated by changes in the competence of multipotent progenitors. Here, we show that a secreted factor, growth and differentiation factor 11 (GDF11), controls the numbers of retinal ganglion cells (RGCs), as well as amacrine and photoreceptor cells, that form during development. GDF11 does not affect proliferation of progenitors-a major mode of GDF11 action in other tissues-but instead controls duration of expression of Math5, a gene that confers competence for RGC genesis, in progenitor cells. Thus, GDF11 governs the temporal windows during which multipotent progenitors retain competence to produce distinct neural progeny.     

Formation of retinal ganglion cell topography during prenatal development

A fundamental feature of the mammalian visual system is the nonuniform distribution of ganglion cells across the retinal surface. To understand the ontogenetic processes leading to the formation of retinal ganglion cell topography, changes in the regional density of these neurons were studied in relation to ganglion cell loss and the pattern of retinal growth in the fetal cat. Midway through the gestation period, the density of these neurons was only two to three times greater in the area centralis than in the peripheral retina, whereas shortly before birth this central-to-peripheral difference was nearly 20-fold. Age-related changes in the ganglion cell distribution were found not to correspond in time or magnitude to the massive loss of ganglion cells that occurs during prenatal development. Rather, the formation of ganglion cell density gradients can be accounted for by unequal expansion of the growing fetal retina-peripheral regions expand more than the central region, thereby diluting the peripheral density of ganglion cells to a greater degree. Nonuniform growth, in conjunction with differential periods of neurogenesis of the different types of retinal cells, appears to be a dominant factor regulating overall retinal topography. These results suggest that the differential regional expansion of the fetal retina underlies the formation of magnification factors in the developing visual system.     

Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein

In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.     

Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury

Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor-β signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.     

astray, a zebrafish roundabout homolog required for retinal axon guidance

As growing retinotectal axons navigate from the eye to the tectum, they sense guidance molecules distributed along the optic pathway. Mutations in the zebrafish astray gene severely disrupt retinal axon guidance, causing anterior-posterior pathfinding defects, excessive midline crossing, and defasciculation of the retinal projection. Eye transplantation experiments show that astray function is required in the eye. We identify astray as zebrafish robo2, a member of the Roundabout family of axon guidance receptors. Retinal ganglion cells express robo2 as they extend axons. Thus, robo2 is required for multiple axon guidance decisions during establishment of the vertebrate visual projection.     

Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis

Segmentation genes control cell identities during early pattern formation in Drosophila. One of these genes, fushi tarazu (ftz), is now shown also to control cell fate during neurogenesis. Early in development, ftz is expressed in a striped pattern at the blastoderm stage. Later, it is transiently expressed in a specific subset of neuronal precursor cells, neurons (such as aCC, pCC, RP1, and RP2), and glia in the developing central nervous system (CNS). The function of ftz in the CNS was determined by creating ftz mutant embryos that express ftz in the blastoderm stripes but not in the CNS. In the absence of ftz CNS expression, some neurons appear normal (for example, the aCC, pCC, and RP1), whereas the RP2 neuron extends its growth cone along an abnormal pathway, mimicking its sibling (RP1), suggesting a transformation in neuronal identity.     

Specific tropism of HIV-1 for microglial cells in primary human brain cultures

Human immunodeficiency virus (HIV) frequently causes neurological dysfunction and is abundantly expressed in the central nervous system (CNS) of acquired immunodeficiency syndrome (AIDS) patients with HIV encephalitis or myelopathy. The virus is found mostly in cells of the monocyte-macrophage lineage within the CNS, but the possibility of infection of other glial cells has been raised. Therefore, the effects of different HIV-1 and HIV-2 strains were studied in primary cultures of adult human brain containing microglial cells, the resident CNS macrophages, and astrocytes. These cultures could be productively infected with macrophage-adapted HIV-1 isolates but not with T lymphocyte-adapted HIV-1 isolates or two HIV-2 isolates. As determined with a triple-label procedure, primary astrocytes did not express HIV gag antigens and remained normal throughout the 3-week course of infection. In contrast, virus replicated in neighboring microglial cells, often leading to their cell fusion and death. The death of microglial cells, which normally serve immune functions in the CNS, may be a key factor in the pathogenesis of AIDS encephalitis or myelopathy.     

Adenosine receptors: autoradiographic evidence for their location on axon terminals of excitatory neurons

Adenosine receptors were made visible on light microscopy by autoradiography with tritiated cyclohexyladenosine. In the cerebellum, adenosine receptors were absent in Weaver mice, which lack granule cells, and were displaced in Reeler mice, which have displacements of granule cells. Thus, adenosine receptors appear to be located on the axon terminals of excitatory granule cells in the cerebellum. Removal of one eye of a rat depleted adenosine receptors in the contralateral superior colliculus, suggesting that the receptors occur on axon terminals of excitatory projections from retinal ganglion cells. The presence of adenosine receptors on excitatory axon terminals may explain synaptic inhibition by adenosine and the behavioral effects of xanthines.     

Bioelectric activity in long-term cultures of spinal cord tissues

Fragments of embryonic spinal cord (human, rat, and chick) can regenerate and differentiate in tissue culture. Complex bioelectric activity evoked by electric stimuli indicates that nerve cells in cultures may maintain, for months in vitro, not only the capacity to propagate impulses along their neurites but also a remarkable degree of functional organization resembling the activity of synaptic networks of the central nervous system.     

Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1

Mononuclear phagocytes (microglia, macrophages, and macrophage-like giant cells) are the principal cellular targets for human immunodeficiency virus-1 (HIV-1) in the central nervous system (CNS). Since HIV-1 does not directly infect neurons, the causes for CNS dysfunction in acquired immunodeficiency syndrome (AIDS) remain uncertain. HIV-1-infected human monocytoid cells, but not infected human lymphoid cells, released toxic agents that destroy chick and rat neurons in culture. These neurotoxins were small, heat-stable, protease-resistant molecules that act by way of N-methyl-D-aspartate receptors. Macrophages and microglia infected with HIV-1 may produce neurologic disease through chronic secretion of neurotoxic factors.     

Surface molecules identify groups of growing axons

Studies on vertebrate and invertebrate species have established that, during development, axons have the ability to choose particular paths over others. The chemical basis of this pathfinding is not clear but biochemical differences between neurons have long been postulated to account for the specificity of neuronal connections. Such subtle molecular differences between different cells in a single tissue are difficult to study with standard biochemical techniques but hybridoma technology has offered a potential solution to this type of problem. This technique has made possible the production of monoclonal antibodies for identifying and characterizing a family of glycoproteins which are expressed on the surface of specific axon bundles during the development of the leech nervous system. The results show that groups of growing axons do indeed carry chemically distinct surface molecules.