Vanja Nagy
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Project title:
Regulation of Neurotrophin Receptor Signaling and Endocytosis during Neuronal Development and Plasticity, Type of grant: 3B Reintegration, Amount. 62,000 EUR
Summary: Endocytosis of ligand-bound receptor tyrosine kinases (RTKs) is essential to control cellular responses by adjusting the level of receptors at the cell surface as well as defining specificity of signalling pathways inside cells. The signalling endosome containing NGF (nerve growth factor) and BDNF (brain derived neurotrophic growth factor) neurotrophin receptors was shown to be essential for the survival of neurons. Dr. Dikic’s group has recently shown that the ubiquitin ligase, Cbl, and its interacting partners (CIN85/CDAP2) control multiple steps in the endocytosis of RTKs and actin reorganization in neurites. Yet, little is known about their physiological relevance in vivo. The importance of Cbl pathways in retrograde transport and signalling of BDNF and NGF receptor complexes (signalosomes) will be analyzed in transgenic mice deficient for Cbl/Cbl-b and CIN85/CD2AP. These mice will also be used to test whether Cbl-linked pathways control neurite outgrowth during development and neuronal plasticity in adulthood. Objective: The major goal of this proposal is to study the molecular mechanisms underlying endocytosis of neurotrophin receptors and their physiological importance in development and neuronal plasticity in vivo. In particular, the interaction between intracellular transport and signalling of neurotrophin-receptor signalling complexes (signalosomes) will be analysed. Biological Significance: Understanding molecular mechanisms underlying synaptic plasticity, the basis of our cognitive abilities, is fundamentally important, since they decline with age, protracted neuropathologies and acute brain injuries. Dr. Dikic and colleagues have described a novel signalling pathway in vitro, which have great implications in central nervous system (CNS) development and maintenance. Classically, neurotrophins and their receptors have only been considered during CNS development, and only recently are being appreciated for their role during normal adult CNS function, such as synaptic activity associated with long-term potentiation (LTP), learning and memory. Thus, very little is known about their function and regulation during these fundamental processes. Here, we propose to elucidate the negative regulation of ligand-bound neurotrophin receptors, during development and adult neural plasticity in vivo. Insight into signalling pathways that might be at play during normal brain development and adult function provides possible novel therapeutic targets, and thus contribute to the overall improvement and quality of human life.
PROJECT DURATION: Sept. 2008-March 2010
PROJECT DESCRIPTION:
UPS is a highly diverse cellular process that controls cellular fate by participating in various signaling pathways, such as protein degradation, DNA repair, receptor internalization, apoptosis, development and metastasis to name a few. Critical components of this system are a series of enzymes (E1, E2 and E3) which cooperate in order to conjugate ubiquitin molecules onto target proteins. Although the idea that active UPS is required for neuronal development and function, including memory formation, is gaining strength very little is known of the specific pathways involved. For this reason we sought to investigate ubiquitination of the neurotrophin receptors during development and neuronal activity. In our efforts to complete this task, we have identified novel E3 ligase interaction in the neuron: sharpin and parkin. Sharpin is found in postsynaptic terminals (PSD) of excitatory neurons where it binds shank (PSD scaffold molecule); however, its biological function is unknown. Understanding the function and biological significance of these novel interacting partners in the synapse with enrich our understanding of neuronal plasticity during development as well as synaptic activity, such as the kind associated with memory formation.
RESULTS:
A yeast two hybrid screen (Y2H) originally identified sharpin to bind to UBL domain of parkin. This was confirmed by HEK transfected cell lines followed by co-immunoprecipitation (co-IP), as well as endogenous co-IP performed in lysates of adult male rat hippocampal tissue. We have determined the domains involved in this interaction with a series of deletion mutations in both proteins. The biological function and significance of this interaction, however, remains unknown. Another Y2H screen was performed to identify interacting partners of sharpin in an effort to further characterize its function. This work was done with a diploma student, Tea Copic, and its results will be written up as part of her thesis.
Immunocytochemical and biochemical analysis of sharpin subcellular localization in both primary neuronal cultures as well as in HEK and Hela cell lines, showed a very strong nuclear localization. Bioinformatical analysis did not reveal a nuclear localization sequence, however, predicted a strong leucine-rich nuclear export sequence (NES). Using chemical and point mutation methods we confirmed the existance of NES in the N-terminal domain of sharpin. Preliminary evidence also suggests that sharpin directly binds DNA. Further analysis is needed to determine the significance of these findings. This aspect of the project was in part performed by Nenad Medic, a diploma student in collaboration with Tomislav Kokotovic, a visiting medical student. Nenad Medic sucessfully defended his diploma thesis on this topic, 21.12.09 titled, „Karakterizacija proteina sharpina“ eng „Characterization of sharpin“.
MILESTONES:
Recruited and trained 3 diploma students:
Nenad Medic, diploma thesis, PMF, Zagreb “Karakterizacija proteina sharpina”, defense Dec. 2009
Tomislav Kokotovic, Medical School, Zagreb, Role of sharpin in the NF-kappaB pathway
Tea Copic, diploma thesis, PMF, Split
“Interakcija E3 ligaze parkina i novootkrivenog proteina sharpina”, defense TBA.
Publications:
- Nagy V, and Dikic I. Ubiquitin ligase complexes: from substrate selectivity to conjugational specificity. Biol Chem. 2010 Feb-Mar;391(2-3):163-9.
- Ikeda F et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Submitted.