Request for Funding
Medical Student Research Fellowship for Summer 2011
All descriptions must contain enough detail to permit an assessment of the problem that is to be addressed and the methodologies that are to be employed. Please be careful to outline the role that the student will play in the project that is described. Please ensure that all relevant approval numbers (IRB, IACUC) are provided.
Mentor: Joseph Albanesi, Ph.D.
Mail Code: 9041
Phone number: 214-645-6119
Project title: Basic Science Research on Arc Palmitoylation
Human subjects IRB approved project number (where applicable):
Animal subjects IRB approved project number (where applicable):
Project Type Basic Research
Brief Description of Project:
Background: Sustained changes in synaptic efficacy, such as those observed during long-term potentiation (LTP) and long-term depression (LTD), are underlying mechanisms of learning and memory. LTP and LTD are accompanied by increases and decreases, respectively, in the expression of AMPA type glutamate receptors (AMPARs) on the plasma membrane of dendritic spines. These changes in surface AMPAR expression are controlled by changes in the balance between exocytosis and endocytosis of AMPAR-containing vesicles in response to neuronal activity. Another key aspect of LTP and LTD is the rapid induction of Immediate Early Genes, including the protein that I will investigate this summer, Activity-regulated cytoskeletal-associated protein (Arc). Arc is a 45 kDa protein that was first isolated from detergent-insoluble cytoskeletal fractions of brain tissue and hippocampal cells. LTD is impaired in Arc knockout mice, which also show a 2-fold increase in surface expression of AMPARs, suggesting that Arc enhances endocytosis of AMPARs from the post-synaptic plasma membrane. In support of this possibility, overexpression of Arc decreases AMPAR surface expression in dissociated hippocampal neurons, and Arc was shown to interact directly with an element of the endocytic machinery, dynamin, a ~100 kDa GTPase that polymerizes around the necks of invaginating vesicles and promotes membrane scission in a GTPase-dependent manner.
Given Arc’s involvement AMPAR internalization, it is not surprising that impairment of cognition has been linked to abnormal elevation in Arc expression. For example, increased Arc levels are associated with two major cognitive disorders, Fragile X syndrome (FXS) and Angelman’s syndrome. FXS is caused by mutations in the FXS mental retardation protein, FMRP, a translational repressor that inhibits the expression of several LTD-linked proteins, including Arc. Angelman’s syndrome is caused by mutations in the gene encoding the E3 ubiquitin ligase, UBE3A, which is responsible for ubiquitylation of Arc and its subsequent degradation by the proteasome.
Proposed research: It is believed that Arc recruits dynamin to the plasma membrane, but the molecular basis for the Arc-membrane interaction is not understood, because Arc is a soluble, highly negatively-charged protein. Recent results from the Albanesi lab indicate that Arc undergoes a post-translational modification known as palmitoylation, the covalent attachment of a 16-carbon palmitoyl chain that serves to anchor certain proteins into the hydrophobic core of the membrane bilayer. The objective of my summer research is to determine if palmitoylation is necessary to target Arc to membranes in living SH-SY5Y cells, which are neuron-like and normally express Arc. I will transfect these cells with a fluorescently tagged form of Arc (EGFP-Arc). A subset of transected cells will be treated with 2-bromopalmitate (2-BP), an inhibitor of the enzymes that catalyze protein palmitoylation. EGFP-Arc will be visualized in control and treated cells by conventional fluorescence microscopy, and also using a method known as TIRF/FFS (Total Internal Reflection Fluorescence/Fluorescence Fluctuation Spectroscopy), which allows one to quantify the number and mobility of fluorescently tagged molecules in the plasma membrane. In parallel, I will be assisting in an analysis aimed at identifying the palmitoylated cysteines in Arc. This will allow us to generate mutants which fail to undergo palmitoylation, with the long-term goal (beyond the scope of my project) of determining the significance of palmitoylation for AMPAR internalization in cultured neurons and in mice
Previous Research Activities or Publications with Medical Students: