Ann M. Turnley

Axonal Regeneration and Lack of Astrocytic Gliosis in EphA4-Deficient Mice

Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (-/-), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4-/- mice recovered stride length, the ability to walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1-3 months of injury. EphA4 expression was upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned EphA4-/- spinal cords. EphA4-/- astrocytes failed to respond to the inflammatory cytokines, interferon-γ or leukemia inhibitory factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4-/- astrocytes, but longer neurites when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury, axonal inhibition, and astrocytic gliosis.

LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival

Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults. Available therapies can inhibit the inflammatory component of MS but do not suppress progressive clinical disability. An alternative approach would be to inhibit mechanisms that drive the neuropathology of MS, which often includes the death of oligodendrocytes, the cells responsible for myelinating the CNS. Identification of molecular mechanisms that mediate the stress response of oligodendrocytes to optimize their survival would serve this need. This study shows that the neurotrophic cytokine leukemia inhibitory factor (LIF) directly prevents oligodendrocyte death in animal models of MS. We also demonstrate that this therapeutic effect complements endogenous LIF receptor signaling, which already serves to limit oligodendrocyte loss during immune attack. Our results provide a novel approach for the treatment of MS.

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system.

Abstract: The mammalian AMP‐activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (α, β, and γ) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the α2 and β2 subunits were increased between embryonic days 10 and 14, whereas expression of α1, β1, and γ1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The α2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the α1 catalytic subunit showed low expression in neuropil. The γ1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the β1 and β2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either β isoform. Preferential nuclear localization of the α2, β1, and γ1 subunits suggests new functions of the AMP‐activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits α1 and α2 point to different physiological roles.

EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Disease onset and progression are variable, with survival ranging from months to decades. Factors underlying this variability may represent targets for therapeutic intervention. Here, we have screened a zebrafish model of ALS and identified Epha4, a receptor in the ephrin axonal repellent system, as a modifier of the disease phenotype in fish, rodents and humans. Genetic as well as pharmacological inhibition of Epha4 signaling rescues the mutant SOD1 phenotype in zebrafish and increases survival in mouse and rat models of ALS. Motor neurons that are most vulnerable to degeneration in ALS express higher levels of Epha4, and neuromuscular re-innervation by axotomized motor neurons is inhibited by the presence of Epha4. In humans with ALS, EPHA4 expression inversely correlates with disease onset and survival, and loss-of-function mutations in EPHA4 are associated with long survival. Furthermore, we found that knockdown of Epha4 also rescues the axonopathy induced by expression of mutant TAR DNA-binding protein 43 (TDP-43), another protein causing familial ALS, and the axonopathy induced by knockdown of survival of motor neuron 1, a model for spinomuscular atrophy. This suggests that Epha4 generically modulates the vulnerability of (motor) neurons to axonal degeneration and may represent a new target for therapeutic intervention.

Interferon-γ but not TNFα promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells

Abstract Neural trauma, such as traumatic brain injury or stroke, results in a vigorous inflammatory response at and near the site of injury, with cytokine production by endogenous glial cells and invading immune cells. Little is known of the effect that these cytokines have on neural stem cell function. Here we examine the effects of two inflammatory cytokines, interferon-γ (IFNγ) and tumour necrosis factor-α (TNFα), on adult neural stem cells. Neural stem cells grown in the presence of either cytokine failed to generate neurospheres. Cytotoxicity assays showed that TNFα but not IFNγ was toxic to the neural stem cells under proliferative conditions. Under differentiating conditions, neither cytokine was toxic; however, IFNγ enhanced neuronal differentiation, rapidly increasing βIII-tubulin positive cell numbers 3–4 fold and inhibiting astrocyte generation. Furthermore, neurite outgrowth and the number of neurites per neuron was enhanced in cells differentiated in the presence of IFNγ. Therefore, both inflammatory cytokines examined have substantial, but different effects on neural stem cell function and suggests that regulation of the inflammatory environment following brain injury may influence the ability of neural stem cells to repair the damage.

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2−/− mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone–mediated downregulation of Ngn1.

Roles of Eph receptors and ephrins in the normal and damaged adult CNS

Injury to the central nervous system (CNS) usually results in very limited regeneration of lesioned axons, which are inhibited by the environment of the injury site. Factors that have been implicated in inhibition of axonal regeneration include myelin proteins, astrocytic gliosis and cell surface molecules that are involved in axon guidance during development. This review examines the contribution of one such family of developmental guidance molecules, the Eph receptor tyrosine kinases and their ligands, the ephrins in normal adult CNS and following injury or disease. Eph/ephrin signaling regulates axon guidance through contact repulsion during development of the CNS, inducing collapse of neuronal growth cones. Eph receptors and ephrins continue to be expressed in the adult CNS, although usually at lower levels, but are upregulated following neural injury on different cell types, including reactive astrocytes, neurons and oligodendrocytes. This upregulated expression may directly inhibit regrowth of regenerating axons; however, in addition, Eph expression also regulates astrocytic gliosis and formation of the glial scar. Therefore, Eph/ephrin signaling may inhibit regeneration by more than one mechanism and modulation of Eph receptor expression or signaling could prove pivotal in determining the outcome of injury in the adult CNS.

Cytokines that Signal Through the Leukemia Inhibitory Factor Receptor-β Complex in the Nervous System

Abstract: Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as “suppressor of cytokine signalling” proteins, which inhibit the JAK/STAT but not the mitogen‐activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.

Regulation of endogenous neural stem/progenitor cells for neural repair - factors that promote neurogenesis and gliogenesis in the normal and damaged brain

Neural stem/precursor cells in the adult brain reside in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. These cells primarily generate neuroblasts that normally migrate to the olfactory bulb (OB) and the dentate granule cell layer respectively. Following brain damage, such as traumatic brain injury, ischemic stroke or in degenerative disease models, neural precursor cells from the SVZ in particular, can migrate from their normal route along the rostral migratory stream (RMS) to the site of neural damage. This neural precursor cell response to neural damage is mediated by release of endogenous factors, including cytokines and chemokines produced by the inflammatory response at the injury site, and by the production of growth and neurotrophic factors. Endogenous hippocampal neurogenesis is frequently also directly or indirectly affected by neural damage. Administration of a variety of factors that regulate different aspects of neural stem/precursor biology often leads to improved functional motor and/or behavioral outcomes. Such factors can target neural stem/precursor proliferation, survival, migration and differentiation into appropriate neuronal or glial lineages. Newborn cells also need to subsequently survive and functionally integrate into extant neural circuitry, which may be the major bottleneck to the current therapeutic potential of neural stem/precursor cells. This review will cover the effects of a range of intrinsic and extrinsic factors that regulate neural stem/precursor cell functions. In particular it focuses on factors that may be harnessed to enhance the endogenous neural stem/precursor cell response to neural damage, highlighting those that have already shown evidence of preclinical effectiveness and discussing others that warrant further preclinical investigation.

Treadmill Training after Spinal Cord Hemisection in Mice Promotes Axonal Sprouting and Synapse Formation and Improves Motor Recovery

Treadmill training with weight-support is a therapeutic strategy used in human patients after spinal cord injury (SCI). Exercise leads to locomotor improvement in a variety of animal models; however, the effect of exercise on axonal regrowth has not been directly examined. This study used several locomotor tests, including kinematic gait analysis, to analyze the differences between treadmill-trained and untrained mice in the usage of their paretic hindlimb following a low thoracic hemisection. Analysis of muscle atrophy, anterograde axonal tracing and expression of the synaptic markers synaptophysin and PSD95 were used to correlate observed behavioural changes with anatomical data. Treadmill trained mice showed significant improvement in use of their paretic hindlimb after 4 weeks of exercise compared to untrained mice in an open field locomotor test (Basso-Beattie-Bresnahan [BBB] scale), grid walking and climbing and inter-limb coordination tests. Movement of their hip joint started to approximate the pattern of intact mice, with concomitant use of their ankle. Unlike untrained mice, exercised mice showed decreased muscle atrophy, increased axonal regrowth and collateral sprouting proximal to the lesion site, with maintenance of synaptic markers on motor neurons in the ventral horn. However, there was no axonal regeneration into or across the lesion site indicating that the improved behaviour may have been, at least in part, due to enhanced neural activity above the lesion site.