Research Interests: Molecular regulation of excitatory synaptic transmission; control of energy metabolism; Synaptic remodeling and neuronal cell death.Research Profile
We examine the mechanism by which activation of N-Methyl-D-Aspartate receptors (NMDARs) regulates the neural specific glucose transporter-3 (GLUT3). Glucose uptake through GLUT3 can influence a number of homeostatic cellular processes including synaptic transmission and cellular energetic. Since neurons are high energy-dependent cell, we hypothesize that neural activity regulates intracellular glucose levels by controlling the expression of cell surface GLUT3. We show that normal activation of NMDARs increases cell surface GLUT3. However, the mechanism by which neural activity regulates GLUT3 trafficking is unknown.
We are testing whether GLUT3 structural motifs interact with the endosomal sorting machinery to regulate the transporter trafficking on dendrites and axons. We are also examine whether GLUT3 trafficking is influenced by dynamic modification of GLUT3 and Glut3 associated proteins through phosphorylation, nitrosylation and O-GlCNAcylation.
Our goal is to gain a molecular view of the interplay between NMDAR signaling and GLUT3 trafficking. A complete elucidation of these mechanisms is critical for understanding GLUT3 function during physiologic and pathologic conditions of the CNS and PNS.
Major lines of research are:
Using our established model of primary neurons in culture, we will examine:
1) Regulation of GLUT3 Trafficking
2) Regulation of GLUT3 trafficking by NO
3) Regulation of GLUT3 trafficking through activation of cGMP/cGK pathway
4) Regulation of surface GLUT3 by excitotoxic glutamate.
To examine receptor trafficking, synaptic remodeling and activity-dependent signaling pathways, we utilize a variety of molecular and biochemical techniques.
- Expression of tagged-GLUT3 cDNA clone, GLUT3 mutants and various other cDNA clones for nNOS, cGMP kinases and other optical reporter for Ca2+ and glucose will be deployed in primary cultured neurons to examine GLUT3 endosomal and endo-membrane vesicular trafficking on dendrites and axon after treatments that induce GLUT3 trafficking in neurons.
- For biochemical analysis neurons will be fractionated to identify subcellular GLUT3 after chemical treatments that induce GLUT3 trafficking to cell surface.
- For analysis of post-translational changes in proteins by phosphorylation, we will use standard methods for kinase and phosphatase assays, and antibodies raised against protein phosphopeptides, including phospho-nNOS antibodies that we have developed ourselves.
- Mass spectroscopy will be used to identify GLUT3 associated proteins and proteins modification by glycosylation, nitrosylation or phosphorylation.
- We will use mice transgenic mutants and protein knock-down techniques to establish the function of GLUT3 and GLUT3 associated proteins that influenced GLUT3 trafficking in vivo.
- Anti-hypertrophic and anti-oxidant effect of beta3-adrenergic stimulation in myocytes requires differential neuronal NOS phosphorylation. Watts VL, Sepulveda FM, Cingolani OH, Ho AS, Niu X, Kim R, Miller KL, Vandegaer K, Bedja D, Gabrielson KL, Rameau G, O'Rourke B, Kass DA, Barouch LA. J Mol Cell Cardiol. May 2, 2013.
- Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthase mediates penile erection.Hurt KJ, Sezen SF, Lagoda GF, Musicki B, Rameau GA, Snyder SH, Burnett AL.Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16624-9.
- Ferreira, J. M., Burnett A., Rameau G.A. Activity-Dependent Regulation of Surface Glucose Transporter-3. J Neurosci. (2011) Feb. 9; 31 (6): 1991-99.
- Charu, M, Sophie, R, Ferreira J, Rameau ,G.A. Fu, J , Ziff, E.B. (2010) Regulation of Synaptic Structure and Function by Palmitoylated AMPA Receptor Binding Protein. Molecular and Cellular Neuroscience 43 (2010) 341-352.
- Rameau, G.A. Tukey, D.S. Garcin-Hosfield, E.D. Misra, C. Titcombe, R.F. Kathri, L. Getzoff, E.D. and Ziff E.B. (2007). Biphasic Coupling of Neuronal Nitric Oxide Synthase Phosphorylation to the NMDA Receptor Regulates AMPA Receptor Trafficking and Neuronal Cell Death. J Neurosci. 2007 Mar 28; 27 (13): 3445-55
- Rameau, G. A. Lin C. Ziff E.B (2004). Bidirectional Regulation of Neuronal Nitric Oxide Synthase Phosphorylation at Serine 847 by the NMDA Receptor. J Biol Chem, 279(14): 14307-14.
- Rameau, G.A. Lin C. Ziff, E.B. NMDA Receptor Regulation of nNOS Phosphorylation and Induction of Neuron Death. Neurobiology of Aging, 24(8): 1125-1135, 2003
- Rameau, G.A. Yukio, A. Lin C. Ziff E.B. (2000). Role of NMDA Receptor Functional Domains in Excitatory Cell Death. Neuropharmacology, (39) 2255-2266,