Man Lab Research

In the brain, the fast excitatory synaptic transmission is usually mediated by glutamate and AMPA type glutamatergic receptors (AMPARs).AMPARs are heteropentermeric ion channels expressed by almost all neurons in the brain.Long-lasting changes in the strength of synaptic transmission, including long-term potentiation and long-term depression, serve as underlying mechanisms for learning and memory, which are expressed via regulation of AMPAR trafficking, localization and synaptic accumulation.It is therefore extremely important to understand the signaling pathways, molecular components and their dynamic interactions involved in AMPAR trafficking and synaptic accumulation.We aim to understand the cellular/molecular mechanisms mediating synaptic plasticity, including Hebbian-type and homeostatic plasticity, particularly the contribution of AMPAR localization, trafficking and protein stability.We also investigate cellular mechanisms involved in neuron morphogenesis and neuronal bioenergy homeostasis.Currently, we work on the following directions:

(1) Homeostatic regulation of AMPA receptor expression at single synapses. In the central nervous system, a neuron receives a large number of inputs from many synaptically connected cells. Therefore, individual synaptic inputs act independently of one another, based on the activity of the presynaptic neurons. Synaptic plasticity is a synapse autonomous event under physiological conditions. A large amount of data has shown that both Hebbian-type synaptic plasticity, such as LTP and LTD, as well as non-Hebbian type homeostatic synaptic plasticity are expressed by alterations in synaptic AMPAR abundance.Given the fact that plasticity is highly synapse specific, investigation of synapse specific, activity-dependent regulation of AMPAR expression will provide crucial insights into our understanding of synapse physiology. Furthermore, homeostatic plasticity has been studied only at the neuronal population level; whether it is expressed at single synapses and how remains elusive.To address these issues, we have set up two experimental paradigms in neuronal culture, in which the activity levels of identifiable single synapses are specifically up- or down-regulated.We have shown that prolonged inhibition of the activity of a single synapse causes homeostatic elevation of AMPAR expression (Hou et al, 2008).Using a different paradigm, we employed the UV-activable LiGlR6 to selectively increase single synaptic activity.We found that at an activated single synapse, AMPAR expression was homeostatically reduced via receptor endocytosis and proteasome-dependent local degradation.Results of these studies indicate for the first time that via synapse autonomous regulation of MPAR expression, homeostatic regulation can occur at single synapses, suggesting the existence of crucial crosstalk between Hebbian-type and homeostatic synaptic plasticity at individual synapses.

(2) Interaction and functional coordination of the sodium pump (Na, K-ATPase, NKA) and AMPARs. During synaptic transmission mediated mainly via AMPARs, a rapid rise and accumulation of sodium in synaptic spines has been observed.The influx of sodium through AMPARs needs to be counterbalanced by the efficient extrusion of sodium by NKA to maintain ionic homeostasis and neuronal activity. Therefore, coordination between AMPAR and NKA activity should be necessary for neuronal functions.We have recently discovered that the sodium pump is colocalized and physically associated with AMPARs at synapses in hippocampal and cortical neurons.Importantly, dysfunction in NKA induces a rapid decrease in cell-surface-localized AMPARs due to enhanced receptor internalization, and a subsequent receptor removal by proteasome-dependent degradation. (Zhang et al, 2009).We have also indentified that accumulation of intracellular sodium plays a key role in triggering AMPAR removal from the cell surface.

   

(3) AMPAR ubiquitination in receptor trafficking and degradation.Ubiquitin is a small 76 amino acid protein ubiquitously expressed in all eukaryotes.Ubiquitin can be covalently conjugated to other proteins (ubiquitination) through a series of reactions catalyzed by three enzymes: E1-E3.The ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-dependent manner, while E3 is the ligase that links ubiquitin to its substrate at lysine residues and determines substrate specificity.Once a single ubiquitin is conjugated to the target protein (monoubiquitination), an internal lysine in ubiquitin itself can be linked to a second ubiquitin and so on to form a ubiquitin chain (polyubiquitination).Ubiquitination of membrane proteins functions as a tag that can be readily recognized by endocytotic machinery, leading to receptor internalization.Polyubiquitinated proteins are often sorted to the proteasome or lysosome for degradation.The ubiquitin-proteasome system (UPS) is enriched in synapses and plays an important role in synaptic function, including synapse development and synaptic plasticity.AMPARs have a relatively rapid turnover rate with a half life of about 30 hrs. The molecular mechanisms that regulate AMPAR stability are not well understood.We found that AMPARs were subjected to ubiquitination at the lysine residues in the GluR1 C-terminal, leading to alterations in receptor trafficking and stability.More importantly, we have identified the E3 ligase for AMPAR ubiquitination.Exactly how ubiquitination regulates AMPAR trafficking remains to be investigated.

(4) Dynamic association of AMPARs with lipid rafts.Plasma membrane proteins, including a variety of receptor proteins, are immersed in a two-dimensional lipid bilayer.A new view formed in recent years is that the cell membrane is not a homogenous structure.Rather, some species of lipids are aggregated laterally in the membrane to form liquid-ordered lipid microdomains termed lipid rafts, which are enriched in glycospingolipids, cholesterol, and GPI-anchored proteins.We have shown that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent.In mature neurons lipid rafts exist on the entire cell-surface and show high mobility.AMPARs co-localize and associate with lipid rafts in the plasma membrane, and their raft localization is regulated by NMDA receptor activity via the NOS-NO pathway.AMPARs insert in the proximity of the surface raft domain, and perturbation of lipid rafts dramatically suppresses AMPAR exocytosis, resulting in a significant reduction of AMPA receptor cell-surface expression.Although lipid raft localization of AMPARs has been reported, our work provides the first piece of evidence indicating that the association is dynamically regulated, with lipid rafts serving as special sites for AMPAR exocytosis (Hou et al, 2008).

(5) Neuronal glutamate transporters in synaptic function and receptor localization.Glutamate mediates most of the synaptic transmission in the brain via activation of the excitatory ionotrophic AMPARs.Due to a lack of extracellular enzymatic degradation, the removal of released glutamate is achieved through the coordinated activities of glutamate transporters, i.e., excitatory amino acid transporters (EAATs).EAATs are distributed in glia that tightly envelop the synaptic clefts, as well as in neurons.Given the fundamental role of glutamatergic activity for higher brain functions, it is of crucial importance to explore the cellular/molecular details pertaining to the roles of EAAT activity in synapse physiology.Indeed, dysfunction of EAATs has been implicated in many diseases, including stroke, ischemia and neurodegenerative conditions such as amyotropic lateral sclerosis (ALS) and Alzheimer’s disease, as well as psychological problems such as schizophrenia and depression.Glutamate has been believed to be removed mainly by glial EAATs; the roles of neuronal EAATs, which are expressed at pre- and postsynaptic domains, remain poorly understood.We are currently investigating neuronal EAATs including their localization, stability, protein-protein interaction and involvement in synaptic function.

 (6) Neuronal polarization and morphorgenesis.Morphological polarization is the most drastic event occurring during early neuronal development.This process sets the foundation for neuronal network wiring, synapse formation and efficient information transfer in the brain.Polarization is initiated by the selective rapid growth of a single neurite which will eventually differentiate into an axon, whereas the remaining sister neurites become dendrites.The impressive rate of axon growth in the establishment of neuronal polarity necessitates highly efficient protein and membrane synthesis as well as extremely active intracellular delivery and trafficking.We are interested in cellular bioenergy homeostasis and its role in neuronal morphogenesis and synaptic function.

 

 

 

 

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