Qingming Hou

Homeostatic regulation of AMPA receptor expression at single hippocampal synapses

Homeostatic synaptic response is an important measure in confining neuronal activity within a narrow physiological range. Whether or not homeostatic plasticity demonstrates synapse specificity, a key feature characteristic of Hebbian-type plasticity, is largely unknown. Here, we report that in cultured hippocampal neurons, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid subtype glutamate receptor (AMPAR) accumulation is increased selectively in chronically inhibited single synapses, whereas the neighboring normal synapses remain unaffected. This synapse-specific homeostatic regulation depends on the disparity of synaptic activity and is mediated by GluR2-lacking AMPARs and PI3-kinase signaling. These results demonstrate the existence of synaptic specificity and the crucial role of AMPAR-gated calcium in homeostatic plasticity in central neurons.

Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking

J. Neurochem. (2011) 119, 27–39.

A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and Ti plasmid.

A sensor protein ChvG is part of a chromosomally encoded two-component regulatory system ChvG/ChvI that is important for the virulence of Agrobacterium tumefaciens. However, it is not clear what genes ChvG regulates or what signal(s) it senses. In this communication, we demonstrate that ChvG is involved in the regulation of acid-inducible genes, including aopB and katA, residing on the circular and linear chromosomes, respectively, and the tumor-inducing (Ti)-plasmid-harbored vir genes, virB and virE. ChvG was absolutely required for the expression of aopB and very important for the expression of virB and virE. However, it was responsible only for the responsiveness of katA and, to a limited extent, the vir genes to acidic pH. ChvG appears to play a role in katA expression by repressing katA at neutral pH. ChvG had no effect on the expression of two genes that were not acid-inducible. Because ChvG regulates unlinked acid-inducible genes encoding different functions in different ways, we hypothesize that ChvG is a global sensor protein that can directly or indirectly sense extracellular acidity. We also analyzed the re-sequenced chvG and found that ChvG is more homologous to its Sinorhizobium meliloti counterpart ExoS than was previously thought. Full-length ChvG is conserved in members of the α-proteobacteria, whereas only the C-terminal kinase domain is conserved in other bacteria. Sensing acidity appears to enable Agrobacterium to coordinate its coping with the environment of wounded plants to cause tumors.

Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death

Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.

Na,K-ATPase Activity Regulates AMPA Receptor Turnover through Proteasome-Mediated Proteolysis

Neuronal activity largely depends on two key components on the membrane: the Na,K-ATPase (NKA) that maintains the ion gradients and sets the foundation of excitability, and the ionotropic glutamatergic AMPA receptors (AMPARs) through which sodium influx forms the driving force for excitation. Because the frequent sodium transients from glutamate receptor activity need to be efficiently extruded, a functional coupling between NKA and AMPARs should be a necessary cellular device for synapse physiology. We show that NKA is enriched at synapses and associates with AMPARs. NKA dysfunction induces a rapid reduction in AMPAR cell-surface expression as well as total protein abundance, leading to a long-lasting depression in synaptic transmission. AMPAR proteolysis requires sodium influx, proteasomal activity and receptor internalization. These data elucidate a novel mechanism by which NKA regulates AMPAR turnover and thereby synaptic strength and brain function.

Regulation of AMPA receptor localization in lipid rafts

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS-NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression.

Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons.

Single-cell genomic analysis has grown rapidly in recent years and finds widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. To date, the amplification bias, amplification uniformity and reproducibility of the three major single cell whole genome amplification methods (GenomePlex WGA4, MDA and MALBAC) have not been systematically investigated using mammalian cells. In this study, we amplified genomic DNA from individual hippocampal neurons using three single-cell DNA amplification methods, and sequenced them at shallow depth. We then systematically evaluated the GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. The MALBAC displays significant biases towards high GC content. We then attempted to correct the GC bias issue by developing a bioinformatics pipeline, which allows us to call CNVs in single cell sequencing data, and chromosome level and sub-chromosomal level CNVs among individual neurons can be detected. We also proposed a metric to determine the CNV detection limits. Overall, MALBAC and WGA4 have better performance than MDA in detecting CNVs.

MicroRNA miR124 is required for the expression of homeostatic synaptic plasticity

Homeostatic synaptic plasticity is a compensatory response to alterations in neuronal activity. Chronic deprivation of neuronal activity results in an increase in synaptic AMPA receptors (AMPARs) and postsynaptic currents. The biogenesis of GluA2-lacking, calcium-permeable AMPARs (CP-AMPARs) plays a crucial role in the homeostatic response; however, the mechanisms leading to CP-AMPAR formation remain unclear. Here we show that the microRNA, miR124, is required for the generation of CP-AMPARs and homeostatic plasticity. miR124 suppresses GluA2 expression via targeting its 3′-UTR, leading to the formation of CP-AMPARs. Blockade of miR124 function abolishes the homeostatic response, whereas miR124 overexpression leads to earlier induction of homeostatic plasticity. miR124 transcription is controlled by an inhibitory transcription factor EVI1, acting by association with the deacetylase HDAC1. Our data support a cellular cascade in which inactivity relieves EVI1/HDAC-mediated inhibition of miR124 gene transcription, resulting in enhanced miR124 expression, formation of CP-AMPARs and subsequent induction of homeostatic synaptic plasticity.

The deubiquitinating enzyme USP46 regulates AMPA receptor ubiquitination and trafficking.

Alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPARs) are the primary mediators for inter‐neuronal communication and play a crucial role in higher brain functions including learning and memory. Our previous work demonstrated that AMPARs are subject to ubiquitination by the E3 ligase Nedd4, resulting in EPS15‐mediated receptor internalization and Ubiquitin (Ub)–proteasome pathway (UPP)‐dependent degradation. Protein ubiquitination is a highly dynamic and reversible process, achieved via the balance between ubiquitination and deubiquitination. However, deubiquitination of mammalian AMPARs and the responsible deubiquitinating enzymes remain elusive. In this study, we identify USP46 as the deubiquitinating enzyme for AMPARs. We find that AMPARs are subject to K63 type ubiquitination, and USP46 is able to deubiquitinate AMPARs in vivo and in vitro. In heterologous cells and neurons, expression of USP46 results in a significant reduction in AMPAR ubiquitination, accompanied by a reduced rate in AMPAR degradation and an increase in surface AMPAR accumulation. By contrast, knockdown of USP46 by RNAi leads to elevated AMPAR ubiquitination and a reduction in surface AMPARs at synapses in neurons. Consistently, miniature excitatory postsynaptic currents recordings show reduced synaptic strength in neurons expressing USP46‐selective RNAi. These results demonstrate USP46‐mediated regulation of AMPAR ubiquitination and turnover, which may play an important role in synaptic plasticity and brain function.

Agrobacterium VirD2-Binding Protein Is Involved in Tumorigenesis and Redundantly Encoded in Conjugative Transfer Gene Clusters

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.