Onderzoek naar het mechanisme en therapeutisch potentieel van het efrinesysteem in ALS

Amyotrophic Lateral Sclerosis (ALS) is a dramatic neurodegenerative disease due to its progressive character, the short survival of the patient and the enormous impact on his/her quality of life and that of his/her caretaker. Interestingly, ALS patients, even carrying the same mutation in SOD1, have great variations in clinical characteristics as age of onset, disease duration and severity of the disease. This indicates that ALS is a multifactorial disease influenced by modifying genes and environmental factors. Epha4 is such a genetic factor that modifies ALS both in animal models as well as in patients. We investigated the mechanism in which Epha4 is modifying the disease pathogenesis in ALS. We found that vulnerable motor neurons have high Epha4 expression and that Epha4 reduced the reinnervating capacity of motor neurons, possibly contributing to the higher vulnerability of Epha4 expressing motor neurons in ALS. Furthermore, deleting the Epha4 cytoplasmic domain did not affect disease onset and survival in the SOD1G93A mouse model indicating that Epha4 forward signaling in the Eph-bearing cell does not play a role in the modifying effect of Epha4 in ALS. We therefore suggest that the reverse pathway in the ephrin-bearing cell should be modified in order to alter ALS pathology. We studied ephrinb2 as a possible ligand for the Epha4-mediated effect in ALS. It was highly expressed in motor neurons and in reactive astrocytes. However, deletion of ephrinb2 enhanced disease rather than attenuating it. Interestingly, deleting ephrinb2 from the astrocytes impaired the blood brain barrier integrity which possibly contributed to the reduced disease duration and survival in the SOD1G93A mouse model. To inhibit Epha4 reverse signaling we developed Nanobodies targeting the EphA4 ligandbinding domain. Nb 39 and Nb 53 were selective for EphA4, and had KD and IC50 values in the nanomolar range. Both Nanobodies were able to block the interaction of EphA4 with all ephrin ligands and they inhibited ephrin-induced phosphorylation. In conclusion, blocking the interaction of EphA4 and its ephrin ligands will be the most efficient way to block EphA4 reverse signaling and to develop a specific EphA4 antagonist.status: publishe