Chaperone-mediated autophagy modulates synaptic differentiation in hippocampal neurons

Espinoza, Claudia.; Hormazabal Juan.; De la Cruz, Javiera.; Cancino, Robert.; Alfaro, Iván E.


Introduction: Development of neuronal synapses is characterized by a coordinated assembly of proteins at pre- and post-synaptic regions. Protein homeostasis is crucial for the proper development, function and plasticity of synapse. Both local protein synthesis and degradation are important for these processes; however the role of selective protein degradation by Chaperone Mediated Autophagy (CMA) in synapse development is unknown. Materials and Methods: We use immunofluorescence of LAMP2A, a protein critically involved in CMA, and a photoactivable fluorescent reporter of CMA to determinate the presence of CMA-active lysosomes in neuronal projections of cultured hippocampal neurons. Additionally, a shRNA mediated knockdown of LAMP2A was used to evaluate the effects of CMA activity in glutamatergic synapse development using microscopy imaging techniques. Results: Using bioinformatics analysis, we identified that 31.9% and 60.2% proteins from the pre- and post-synaptic proteome contain CMA-targeting KFERQ motifs, respectively. LAMP2A expression increases in the period of synapse formation and reaches a peak during late stages of neuronal differentiation, being in close proximity to glutamatergic synapses. Moreover, a CMA fluorescent reporter becomes compartmentalized in discrete domains in neurites indicative of the presence of CMA active lysosomes in neurites of hippocampal neurons. On the other hand, shRNA mediated knockdown of LAMP2A induces a significant increase in the density of synaptic contacts and changes in synaptic morphology increasing the size of clusters of the postsynaptic protein PSD95. Discussion: In summary, these results indicate that CMA is present at neurites and modulates synaptic formation negatively. Additional experiments are in progress to determine if CMA limit the formation of new synaptic contacts or participates in the elimination of immature synapses.Acknowledgements: Fondecyt 11161056, Proyecto FCPV Apoyo Basal AFB170004.

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Fecha de publicación: 2018
Año de Inicio/Término: 18 de Octubre de 2018