Yona Goldshmit

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.

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.

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.

Fgf-Dependent Glial Cell Bridges Facilitate Spinal Cord Regeneration in Zebrafish

Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this “glial bridge” and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.

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.

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice.

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.

Retinal afferents synapse with relay cells targeting the middle temporal area in the pulvinar and lateral geniculate nuclei.

Considerable debate continues regarding thalamic inputs to the middle temporal area (MT) of the visual cortex that bypass the primary visual cortex (V1) and the role they might have in the residual visual capability following a lesion of V1. Two specific retinothalamic projections to area MT have been speculated to relay through the medial portion of the inferior pulvinar nucleus (PIm) and the koniocellular layers of the dorsal lateral geniculate nucleus (LGN). Although a number of studies have demonstrated retinal inputs to regions of the thalamus where relays to area MT have been observed, the relationship between the retinal terminals and area MT relay cells has not been established. Here we examined direct retino-recipient regions of the marmoset monkey (Callithrix jacchus) pulvinar nucleus and the LGN following binocular injections of anterograde tracer, as well as area MT relay cells in these nuclei by injection of retrograde tracer into area MT. Retinal afferents were shown to synapse with area MT relay cells as demonstrated by colocalization with the presynaptic vesicle membrane protein synaptophysin. We also established the presence of direct synapes of retinal afferents on area MT relay cells within the PIm, as well as the koniocellular K1 and K3 layers of the LGN, thereby corroborating the existence of two disynaptic pathways from the retina to area MT that bypass V1.

Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness.

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling‐2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper‐responsive SOCS2 null (−/−) mice and GH non‐responsive GH receptor null (GHR−/−) mice. While SOCS2−/− mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR−/− mice showed a 25% increase even though brain size was decreased. Interneuron sub‐populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2−/− mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR−/− mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron : glia ratio was increased in GHR−/− mice and decreased in SOCS2−/− mice. The astrocytes in GHR−/− mice appeared smaller, while they were larger in SOCS2−/− mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR−/− mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2−/− neurons were larger than GHR−/− neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR−/− and SOCS2−/− mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.

Species-specific microRNA roles elucidated following astrocyte activation

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in regulation of gene expression by binding to target genes. Many miRNAs were associated with the function of the central nervous system (CNS) in health and disease. Astrocytes are the CNS most abundant glia cells, providing support by maintaining homeostasis and by regulating neuronal signaling, survival and synaptic plasticity. Astrocytes play a key role in repair of brain insults, as part of local immune reactivity triggered by inflammatory or pathological conditions. Thus, astrocyte activation, or astrogliosis, is an important outcome of the innate immune response, which can be elicited by endotoxins such as lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-γ). The involvement of miRNAs in inflammation and stress led us to hypothesize that astrogliosis is mediated by miRNA function. In this study, we compared the miRNA regulatory layer expressed in primary cultured astrocyte derived from rodents (mice) and primates (marmosets) brains upon exposure to LPS and IFN-γ. We identified subsets of differentially expressed miRNAs some of which are shared with other immunological related systems while others, surprisingly, are mouse and rat specific. Of interest, these specific miRNAs regulate genes involved in the tumor necrosis factor-alpha (TNF-α) signaling pathway, indicating a miRNA-based species-specific regulation. Our data suggests that miRNA function is more significant in the mechanisms governing astrocyte activation in rodents compared to primates.

ErbB-4 activation inhibits apoptosis in PC12 cells.

Neuregulins, a large family of polypeptide growth factors, exert various distinctive effects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced differentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)alpha treatment. The neuregulin-induced survival is comparable to the effect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNF alpha treatment. Moreover, neuregulin like NGF induces the survival of neuronal differentiated PC12-ErbB-4 cells. The survival effect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulin-induced survival effect. Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks.