Study of the Role of Plasmin- mediated Cleavage of Erythropoietin- Producing Hepatocellular A4 Receptor and its Molecular Binding Partners in Anxiogenesis

Stressful experiences can produce a variety of emotional states, including elevated and prolonged levels of anxiety that can lead to anxiety disorders. Anxiety disorders are the most prevalent class of mental disorders worldwide; however, the neurobiological mechanisms that regulate anxiety and its disorders are still not well understood. In this context, the current project delves into downstream effects of the cleavage of EphA4 receptor by tissue plasminogen activator (tPA)/plasmin proteases; an event recently discovered by former members of our laboratory. The initial hypothesis of this project was, therefore, that tPA/plasmin-induced proteolysis of the murine EphA4 receptor is present in the mouse brain and can regulate stress-related phenomena. An initial necessary first step was to confirm the presence of the proteolytic cascade in areas relevant for the study of anxiety. In agreement with this hypothesis, I demonstrated that tPA and plasminogen co-localise with EphA4 in the GABAergic neuronal synapses of the central amygdala (CeA) through immunhistochemical techniques. In line with this discovery, the relevant literature sufficiently proves that inhibitory interneurons in the central amygdala of the mouse brain regulate anxiety-related processes by controlling the activity of downstream output cells. Specifically, those of the lateral subdivision of the central amygdala (CeL) expressing protein kinase C delta (PKCδ+) are important for aversive stimuli processing and memory. In the present work, I show that all tPA-expressing cells in CeA are also PKCδ+, which establishes a strong link between PKCδ+ cell-types and the location of an assumptive tPA/ plasmin/EphA4 cascade in areas relevant for stress-related events and anxiety- like behaviours. Conceivably, PKCδ+ (tPA) cells can regulate the properties of their downstream GABAergic synapses during stress through a cleavage of EphA4 associated with the tPA/plasmin proteolytic cascade. It is known that stressful stimuli produce the tPA-mediated conversion of plasminogen into the active enzyme, plasmin; and, as demonstrated here, plasmin would subsequently be able to cleave the tyrosine kinase receptor, EphA4. Cleavage of EphA4 has multiple neurobiological consequences. At the molecular level, I examined how shedding of EphA4 affects postsynaptic GABAergic protein-protein interactions. Here, I show that cleavage induces the dissociation of EphA4 from the GABA-receptor anchoring protein, gephyrin. The repercussions of this event are still unknown. Furthermore, this shedding can regulate the dendritic spine shape as evidenced by spine morphology experiments. Spine morphology is thought to reflect the strength and activity of a synapse whereby the excitatory or inhibitory tone of a neuron can be tuned. Moreover, consistent with a crucial role of the tPA/plasmin/EphA4 signalling cascade in anxiogenesis, EphA4 main cleaved form of EphA4 is increased after restraint stress. Accordingly, increased protein levels in the central amygdala of a plasmin-resistant variant of EphA4 (crEphA4) prevents the expression of stress-induced anxiety-like behaviours in mice, whereas the expression of a truncated EphA4 variant that mimics the cleavage by plasmin (tEphA4) increases this expression. This indicates that the cleavage of EphA4 potentially helps to modulate the expression of anxiety-like behaviours. Therefore, the present work identified a central molecular cascade that potentially controls the structure and function of GABAergic synapses downstream of CeL-PKCδ+ interneurons in the CeA and has the ability to modify the expression of anxiety-like behaviours. Additional pieces of data presented in this work indicate that the cleavage of EphA4 is affected in other brain conditions in which tPA/ plasmin cascade is involved, such as rodent models of stroke or epilepsy. Therefore, this work opens future possibilities for the study of other mechanisms regulated by tPA/plasmin/EphA4 cascade.European Commissio