Final Report Summary - ENDOAMPAR (Endosomal sorting of AMPA receptors for synaptic plasticity)
Executive Summary:
Overall, the main objective of the proposal was to elucidate how the endosomal network operates in the highly specialized environment of the synaptic terminal, and how specific molecular components are recruited during the process of synaptic plasticity. For this aim, we developed a variety of molecular tools and the research fellow learnt the basis of electrophysiological recordings (http://web4.cbm.uam.es/joomla-rl/index.php/en/scientific-departments/molecular-neurobiology?id=%20451(si apre in una nuova finestra)).
One of the components of the endosomal network is APPL1 (for Adaptor protein containing PH domain, PTB domain and Leucine zipper motif). Indeed, APPL1-containing vesicles are “specialized endocytic station” which would be assembled at an early stage during Rab5-driven internalization, and would be involved in signalling transduction to the nucleus. We then decided to study whether this endocytic station was present in neurons and had a role in synaptic plasticity.
So far, we have found that APPL1 is expressed in hippocampal neurons and it presents an endosomal-like expression in dendrites and spines. We designed hairpin RNA to block the expression of APPL1 in our hippocampal slices to study whether APPL1 is involved in basal synaptic transmission and/or synaptic plasticity. In addition, we have been able to develop the protein “replacing” technology, in which endogenous protein is replaced by a fluorescence-tagged recombinant protein. This will allow us to express proteins at almost physiological levels and we will be able to monitor trafficking thanks to the fluorescent tag. Using electrophysiological recordings, we concluded that APPL1 is not involved in constitutive recycling of glutamate receptors, since neurons depleted of APPL1 do not show a decrease in synaptic transmission at basal conditions. However, we found that in certain synaptic plasticity models, APPL1 plays an important role.
On one hand we describe an unexpected role of APPL1 on protein trafficking. It has been shown the role of APPL1 in regulated removal of receptors from the plasma membrane in other cell types. We found the opposite action of APPL1. Neurons lacking APPL1 are able to express long-term depression (LTD), which is associated to an endocytic process. However, these neurons are not able to express long-term potentiation, which is associated to a delivery of glutamate receptors to the membrane. We are currently investigating the mechanism of action of APPL1 in this process. So far, we have found that APPL1 is involved in the PI3K pathway transduction during LTP.
On the other hand, when we induced depression by activating metabotropic glutamate receptors (mGluRs), we found that neurons depleted in APPL1 presented less depression than normal neurons. This effect was probably mediated by the Akt pathway, since knocking-down APPL1 reduced the phosphorylation of Akt mediated by the activation of mGluRs. These results were presented in the previous scientific report and are still under investigation by a master student in Dr. José A. Esteban’s Lab.
We would be describing a new model of protein regulation of synaptic plasticity events, in which endosomal proteins, such as APPL1 work as signalling transducers. We are convinced that the endosomal network plays and important role in the regulation of neuronal function. In addition, APPL1 is also involved in Lowe Syndrome (LS), a very rare disease that is caused by mutations in the OCRL (OculoCerebroRenal Lowe disease) protein. All the mutations described affect the APPL1 binding region, suggesting an important role of APPL1 in OCRL localization and/or function. That will be the IRG fellow’s new research line in the future (please see the logo of the project and https://f4r.org/web/projects/view/2(si apre in una nuova finestra)).
Thanks to the Marie Curie Reintegration Grant, the research fellow got independence in her research work and in external collaborations. Also, it allowed her to attend prestigious international meetings to present our results. Without any doubt, the IRG is highly necessary to increase the perspectives for a long-term career.
