Design of Plasmonic Probes for Evaluating the role of PKD1 in the Distribution of Neuronal Glutamate Receptors.

FRAIRE, JC; MASSERONI, MARÍA LUJÁN; JAUSORO, IGNACIO; PERASSI, E; DIAZ AÑEL, A. M.; CORONADO, EDUARDO

Córdoba

Congreso; 16th International Congress on Photobiology (ICP 2014); 2014

International Union of Photobiology

The missorting of metabotropic Glutamate Receptor 1a (mGluR1a) is associated to ischemia, brain trauma, epilepsy, multiple sclerosis, amyothropic lateral sclerosis, and Huntinton and Parkinson diseases.[1,2] In the present work, the distribution of mGluR1a density on neuron cells on subcellular length scales (dendrites and axons) is determinated by evaluating the role played by the protein kinase D1 (PKD1) in the trafficking of membrane proteins comparing the distribution of mGluR1a for endogenous PKD1 expression with experiments performed in the presence of kinase-inactive protein kinase D1 (PKD1-kd). The distribution of mGluR1a was evaluated using Au nanoparticles (NPs) probes specifically functionalized which allow not only to image NPs where this receptor is present but also to quantify by optical means the NP density. This is so because the NP plasmon coupling generates surface enhancement Raman (SERS) response that depends on NP density . This feauture, facilitated a spatial mapping of the mGluR1a density distribution on subcellular length scales. The measured  values were found to be significantly higher on dendrites than on axons for endogenous PKD1, and a significant increase of mGluR1a on axons was observed when PKD1 is altered. A detailed characterization of the spatial distribution of the NP immunolabels through scanning electron microscopy (SEM) confirmed the findings of the all-optical studies (fluorescence bright analysis and dark-field mycroscopy) and provided additional structural details. From the molecular biology poin of view, it is shown that in cultured hippocampal pyramidal cells, mGluR1a is predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive protein kinase D1 (PKD1-kd) dramatically and selectively alter the intracellular trafficking and membrane delivery of mGluR1a containing vesicles. After PKD1 suppression, dendritic membrane proteins are mispackaged into vesicles that are distributed to both axons and dendrites. These results reinforces the idea that in neurons PKD1 regulates the sorting of dendritic proteins and hence has a role in neuronal polarity.