Lewis R

References (3)

Title : A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2 - Kang_2014_J.Biol.Chem_289_29350
Author(s) : Kang Y , Ge Y , Cassidy RM , Lam V , Luo L , Moon KM , Lewis R , Molday RS , Wong RO , Foster LJ , Craig AM
Ref : Journal of Biological Chemistry , 289 :29350 , 2014
Abstract : Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His6-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His6-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.
ESTHER : Kang_2014_J.Biol.Chem_289_29350
PubMedSearch : Kang_2014_J.Biol.Chem_289_29350
PubMedID: 25190809

Title : Coordinated increase in inhibitory and excitatory synapses onto retinal ganglion cells during development - Soto_2011_Neural.Dev_6_31
Author(s) : Soto F , Bleckert A , Lewis R , Kang Y , Kerschensteiner D , Craig AM , Wong RO
Ref : Neural Dev , 6 :31 , 2011
Abstract : BACKGROUND: Neuronal output is shaped by a balance of excitation and inhibition. How this balance is attained in the central nervous system during development is not well understood, and is complicated by the fact that, in vivo, GABAergic and glycinergic synaptogenesis precedes that of glutamatergic synapses. Here, we determined the distributions of inhibitory postsynaptic sites on the dendritic arbors of individual neurons, and compared their developmental patterns with that of excitatory postsynaptic sites. We focused on retinal ganglion cells (RGCs), the output neurons of the retina, which receive excitatory input from bipolar cells and inhibitory input from amacrine cells. To visualize and map inhibitory postsynaptic sites, we generated transgenic mice in which RGCs express fluorescently tagged Neuroligin 2 (YFP-NL2) under the control of the Thy1 promoter. By labeling RGC dendrites biolistically in YFP-NL2-expressing retinas, we were able to map the spatial distribution and thus densities of inhibitory postsynaptic sites on the dendritic arbors of individual large-field RGCs across ages. RESULTS: We demonstrate that YFP-NL2 is present at inhibitory synapses in the inner plexiform layer by its co-localization with gephyrin, the gamma2 subunit of the GABAA receptor and glycine receptors. YFP-NL2 puncta were apposed to the vesicular inhibitory transmitter transporter VGAT but not to CtBP2, a marker of presynaptic ribbons found at bipolar cell terminals. Similar patterns of co-localization with synaptic markers were observed for endogenous NL2. We also verified that expression of YFP-NL2 in the transgenic line did not significantly alter spontaneous inhibitory synaptic transmission onto RGCs. Using these mice, we found that, on average, the density of inhibitory synapses on individual arbors increased gradually until eye opening (postnatal day 15). A small centro-peripheral gradient in density found in mature arbors was apparent at the earliest age we examined (postnatal day 8). Unexpectedly, the adult ratio of inhibitory/excitatory postsynaptic sites was rapidly attained, shortly after glutamatergic synaptogenesis commenced (postnatal day 7). CONCLUSION: Our observations suggest that bipolar and amacrine cell synaptogenesis onto RGCs appear coordinated to rapidly attain a balanced ratio of excitatory and inhibitory synapse densities prior to the onset of visual experience.
ESTHER : Soto_2011_Neural.Dev_6_31
PubMedSearch : Soto_2011_Neural.Dev_6_31
PubMedID: 21864334

Title : A tiered approach to systemic toxicity testing for agricultural chemical safety assessment - Doe_2006_Crit.Rev.Toxicol_36_37
Author(s) : Doe JE , Boobis AR , Blacker A , Dellarco V , Doerrer NG , Franklin C , Goodman JI , Kronenberg JM , Lewis R , McConnell EE , Mercier T , Moretto A , Nolan C , Padilla S , Phang W , Solecki R , Tilbury L , van Ravenzwaay B , Wolf DC
Ref : Crit Rev Toxicol , 36 :37 , 2006
Abstract : A proposal has been developed by the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) for an improved approach to assessing the safety of crop protection chemicals. The goal is to ensure that studies are scientifically appropriate and necessary without being redundant, and that tests emphasize toxicological endpoints and exposure durations that are relevant for risk assessment. The ACSA Systemic Toxicity Task Force proposes an approach to systemic toxicity testing as one part of the overall assessment of a compound's potential to cause adverse effects on health. The approach is designed to provide more relevant data for deriving reference doses for shorter time periods of human exposure, and includes fewer studies for deriving longer term reference doses-that is, neither a 12-month dog study nor a mouse carcinogenicity study is recommended. All available data, including toxicokinetics and metabolism data and life stages information, are taken into account. The proposed tiered testing approach has the potential to provide new risk assessment information for shorter human exposure durations while reducing the number of animals used and without compromising the sensitivity of the determination of longer term reference doses.
ESTHER : Doe_2006_Crit.Rev.Toxicol_36_37
PubMedSearch : Doe_2006_Crit.Rev.Toxicol_36_37
PubMedID: 16708694