Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the brainstem, spinal cord and motor cortex. ALS is characterized by genetic and clinical heterogeneity, suggesting the existence of genetic factors that modify the phenotypic expression of the disease. We previously identified the axonal guidance EphA4 receptor, member of the Eph-ephrin system, as an ALS disease-modifying factor. EphA4 genetic inhibition rescued the motor neuron phenotype in zebrafish and a rodent model of ALS. Preventing ligands from binding to the EphA4 receptor also successfully improved disease, suggesting a role for EphA4 ligands in ALS. One particular ligand, ephrin-A5, is upregulated in reactive astrocytes after acute neuronal injury and inhibits axonal regeneration. Moreover, it plays a role during development in the correct pathfinding of motor axons towards their target limb muscles. We hypothesized that a constitutive reduction of ephrin-A5 signalling would benefit disease progression in a rodent model for ALS. We discovered that in the spinal cord of control and symptomatic ALS mice ephrin-A5 was predominantly expressed in neurons. Surprisingly, reduction of ephrin-A5 levels in SOD1 G93A mice accelerated disease progression and reduced survival without affecting disease onset, motor neuron numbers or innervated neuromuscular junctions in symptomatic mice. These findings suggest ephrin-A5 as a modifier of disease progression that might play a role in the later stages of the disease. Similarly, we identified a more aggressive disease progression in patients with lower ephrin-A5 protein levels in the cerebrospinal fluid without modifying disease onset. In summary, we identified reduced expression of ephrin-A5 to accelerate disease progression in a mouse model of ALS as well as in humans. Combined with our previous findings on the role of EphA4 in ALS our current data suggests different contribution for various members of the Eph-ephrin system in the pathophysiology of a motor neuron disease. Electronic supplementary material The online version of this article (10.1186/s40478-019-0759-6) contains supplementary material, which is available to authorized users.

Reducing EphA4 before disease onset does not affect survival in a mouse model of Amyotrophic Lateral Sclerosis

“…In summary, it may be that other Eph receptors and/or EphA4 ligands could be involved in the pathophysiology of ALS. We have recently shown that a reduction of ephrin-A5 levels to 50% does not improve but accelerates disease progression in the SOD1 G93A mouse model 28 . Whether this effect is caused by inhibitory interactions in cis with EphA4 expressed at the same membrane, or by interactions with other Eph receptors, is still unknown.…”

“…Different mouse strains were obtained at the Jackson Laboratory (Ben Harbor, ME): the human mutant SOD1 overexpressing mouse (B6.SOD1 G93A ; B6.Cg-Tg(SOD1*G93A)1Gur/J; stock number 004435), an EphA4 floxed mutant mouse that has loxP sites flanking the exon 3 of the Epha4 gene (EphA4 tm1.1Bzh ; Epha4tm1.1Bzh/J; stock number 012916), the CAG-CreER TM mouse (B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J; stock number 004682), and the Thy1-CreER T2 mouse (SLICK-H; Tg(Thy1-cre/ERT2,-EYFP)HGfng/PyngJ; stock number 012708). All animals were housed under standard conditions according to the guidelines of the University of Leuven (KU Leuven), with a 12h light-dark cycle and with access to food and water ad libitum as previously described 28 . Mice were maintained in a C57BL/6J genetic background, and were bred between them to obtain the experimental groups: EphA4 F/+ (EphA4 het ), CAG-CreER TM EphA4 F/+ (CAG-EphA4 het ), EphA4 F/F (EphA4), CAG-CreER TM EphA4 F/F (CAG-EphA4), Thy1-CreER T2 EphA4 F/F (Thy1-EphA4), SOD1 G93A EphA4 F/+ (SOD1-EphA4 het ), SOD1 G93A CAG-CreER TM EphA4 F/+ (SOD1-CAG-EphA4 het ), SOD1 G93A EphA4 F/F (SOD1-EphA4), SOD1 G93A CAG-CreER TM EphA4 F/F (SOD1-CAG-EphA4), SOD1 G93A Thy1-CreER T2 EphA4 F/F (SOD1-Thy1-EphA4).…”

“…Mice were maintained in a C57BL/6J genetic background, and were bred between them to obtain the experimental groups: EphA4 F/+ (EphA4 het ), CAG-CreER TM EphA4 F/+ (CAG-EphA4 het ), EphA4 F/F (EphA4), CAG-CreER TM EphA4 F/F (CAG-EphA4), Thy1-CreER T2 EphA4 F/F (Thy1-EphA4), SOD1 G93A EphA4 F/+ (SOD1-EphA4 het ), SOD1 G93A CAG-CreER TM EphA4 F/+ (SOD1-CAG-EphA4 het ), SOD1 G93A EphA4 F/F (SOD1-EphA4), SOD1 G93A CAG-CreER TM EphA4 F/F (SOD1-CAG-EphA4), SOD1 G93A Thy1-CreER T2 EphA4 F/F (SOD1-Thy1-EphA4). For the mice carrying the SOD1 G93A overexpression, only those, whose father had a lifespan shorter than 165 days, were used for breeding and for experimental purposes as previously described 28 . Both males and females were used in every experiment.…”

“…All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, and all animal experiments were approved by the local ethical committee of the KU Leuven (P229/2013 and P229/2017) and conducted in a blinded manner as previously described 28 .…”

“…Disease phenotype monitoring was performed according to guidelines 30 , and as previously described 28 . Briefly, motor coordination was assessed with the hanging wire test, which records the ability of mice to hang upside down from an elevated grid.…”