Date of Award

2013-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Miranda

Abstract

Glycine is one of the major inhibitory neurotransmitters in the central nervous system. It is implicated in the regulation of motor-sensory function such as; pain perception, reflex responses, and is essential for early development of the CNS. In addition to its inhibitory actions, it acts as an obligatory co-agonist for the activation of glutamatergic NMDARs, which are of pivotal importance in the process of learning and memory, its dysfunction leads to several neurodegenerative diseases. Two high affinity Na+/Cl- dependent transporters GlyT1 and GlyT2 tightly regulate the availability of glycine present at the synapse for the activation of NMDARs and GlyRs. GlyT1 is ubiquitously expressed throughout the brain, and is the only GlyT that is present at both glycinergic and glutamatergic synapses. The ability of this protein in the regulation of inhibitory and excitatory synapses has increased the interest on the study of mechanisms that might regulate this transporter, and could be potential targets for treating several neurological disorders. The overall goal of this present work was to investigate the regulation mechanism of GlyT1, exerted by PKC-dependent ubiquitination. We have identified that PKC activation leads to ubiquitination and internalization of GlyT1. The dependency of this effect on PKC-activation was demonstrated by the prevention of ubiquitination and internalization of GlyT1 with the incubation of BIM, a specific PKC-inhibitor prior to PMA stimulation. Once internalized, the transporter can follow two pathways - recycling and degradation - since co-localization with both Rab11 and Rab7 markers for recycling and late endosomes was observed. By using site-directed mutagenesis we observed that ubiquitination can modify any available lysine residue present at either the N- or C- terminals of GlyT1, and this post-translational modification is essential for the internalization of the transporter. We also showed that preventing GlyT1 ubiquitination does not inhibit phosphorylation of the transporter, but it does reverse the down-regulation in glycine uptake caused by PKC activation, which was previously attributed to PKC-phosphorylation. Our study also comprises the identification of PKC; isoform as responsible for the ubiquitination and internalization of GlyT1. Using specific isoform inhibitors for PKC and short interference RNA we identified PKC; as the PKC isoform regulating both ubiquitination and internalization of the transporter. We also aimed to identify the E3-ligase that mediated ubiquitination of GlyT1 in response to PKC activation, based on previous evidence that suggests other transporters such as CAT-1, DAT and EAAT1 ubiquitination is mediated by Nedd4-1 and Nedd4-2, respectively. We performed short interference RNA experiments of these two ligases on PAE cells expressing GlyT1. However, siRNA of Nedd4-1, Nedd4-2, or both did not show any inhibitory effect on the ubiquitination and internalization of GlyT1. This study provides new perspectives for the molecular mechanisms regulating GlyT1.

Language

en

Provenance

Received from ProQuest

File Size

89 pages

File Format

application/pdf

Rights Holder

Susana Barrera

Download

Share

COinS