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.

Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (Ϫ/Ϫ), 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. EphA4Ϫ/Ϫ 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 EphA4Ϫ/Ϫ spinal cords. EphA4Ϫ/Ϫ astrocytes failed to respond to the inflammatory cytokines, interferon-␥ or leukemia inhibitory factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4Ϫ/Ϫ 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.

Abstract:The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (␣, ␤, and ␥) 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 ␣2 and ␤2 subunits were increased between embryonic days 10 and 14, whereas expression of ␣1, ␤1, and ␥1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The ␣2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the ␣1 catalytic subunit showed low expression in neuropil. The ␥1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the ␤1 and ␤2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either ␤ isoform. Preferential nuclear localization of the ␣2, ␤1, and ␥1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits ␣1 and ␣2 point to different physiological roles.