The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons

Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks

Abstract Background Age-related macular degeneration (AMD) is a leading cause of blindness that affects the central region of the retinal pigmented epithelium (RPE), choroid, and neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD leads to progressive retinal degeneration, and in advanced cases, irreversible vision loss. Although genetic analysis, animal models, and cell culture systems have yielded important insights into AMD, the molecular pathways underlying AMD's onset and progression remain poorly delineated. We sought to better understand the molecular underpinnings of this devastating disease by performing the first comparative transcriptome analysis of AMD and normal human donor eyes. Methods RPE-choroid and retina tissue samples were obtained from a common cohort of 31 normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles were generated for macular and extramacular regions, and statistical and bioinformatic methods were employed to identify disease-associated gene signatures and functionally enriched protein association networks. Selected genes of high significance were validated using an independent donor cohort. Results We identified over 50 annotated genes enriched in cell-mediated immune responses that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine learning model and a second donor cohort, we show that the top 20 global genes are predictive of AMD clinical diagnosis. We also discovered functionally enriched gene sets in the RPE-choroid that delineate the advanced AMD phenotypes, neovascular AMD and geographic atrophy. Moreover, we identified a graded increase of transcript levels in the retina related to wound response, complement cascade, and neurogenesis that strongly correlates with decreased levels of phototransduction transcripts and increased AMD severity. Based on our findings, we assembled protein-protein interactomes that highlight functional networks likely to be involved in AMD pathogenesis. Conclusions We discovered new global biomarkers and gene expression signatures of AMD. These results are consistent with a model whereby cell-based inflammatory responses represent a central feature of AMD etiology, and depending on genetics, environment, or stochastic factors, may give rise to the advanced AMD phenotypes characterized by angiogenesis and/or cell death. Genes regulating these immunological activities, along with numerous other genes identified here, represent promising new targets for AMD-directed therapeutics and diagnostics. Please see related commentary: http://www.biomedcentral.com/1741-7015/10/21/abstrac

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons

Mistargeting of Stx3 does not alter polarized localization of cytoskeletal proteins MAP2 (somatodendritic) or Tau (axonal).

<p>(A) Endogenous MAP2 was labeled in neurons (DIV 4) 2 days after cotransfection with Stx3, Stx3Δ38 or Stx4 together with GFP. (B) Quantification of the axon:dendrite polarity index for endogenous MAP2 and Stx constructs. MAP2 was polarized to the somatodendritic region, and was not significantly different between groups, whereas Stx3 was primarily axonal, Stx3Δ38 was throughout the neurons, and Stx4 was primarily somatodendritic. (C) Endogenous Tau was labeled in neurons (DIV 5) 2 days after cotransfection with Stx3, Stx3Δ38 or Stx4 together with GFP. (D) Quantification of the axon:dendrite polarity index for endogenous Tau. Tau was strongly polarized to the axon, and was not significantly different between groups. Bar, 20 μm.</p

The N-terminal FMDE motif of Stx3 is required for axonal targeting.

<p>(A) Stx3 wild-type and mutant constructs are schematically depicted. Tandem myc epitope tags are present at the C-terminus (extracellular domain) to facilitate surface labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163671#pone.0163671.ref023" target="_blank">23</a>]. Stx3Δ38 contains a 38 amino acid N-terminal truncation. In Stx3_AAA, three residues of the <u>F</u>M<u>DE</u> motif of Stx3 were changed to alanine as indicated by ‘AAA’. (B) Mutant Stx3 constructs lacking the FMDE motif are mistargeted to the somatodendritic, as well as axonal plasma membrane. Neurons were coinfected with adenoviruses expressing Stx3Δ38 or Stx3_AAA and GFP, and were live surface labeled with anti-myc antibody (red). Arrowheads denote axon. Bar, 20 μm. (C) Quantification of axon:dendrite polarity index for surface (red bars) and internal (black bars) Stx3, Stx3Δ38 and Stx4 in mature hippocampal neurons. PI ratios for Stx3Δ38 and Stx4 were significantly reduced compared to Stx3. Error bars, SEM; N = 14–18; * P < 0.0001.</p

Mistargeting of Stx3 disrupts axonal polarization of NgCAM and neurexin 1 but not dendritic polarization of transferrin receptor.

<p>(A) NgCAM is targeted to the axon surface when expressed alone (with tdTomato) or in the presence of wild type Stx3, but when cotransfected with Stx3Δ38 or Stx3_AAA, NgCAM is mislocalized to the somatodendritic region together with mutant syntaxin. Neurons were cotransfected with NgCAM-GFP in the absence or presence of Stx3, Stx3Δ38, Stx3_AAA, or Stx4. After overnight expression, the cells were surface labeled for plasma membrane NgCAM (8D9 antibody), and then were permeabilized and internally labeled with anti-myc to detect total syntaxin, and anti-GFP to label total NgCAM. Arrows indicate dendrite, arrowheads denote axon; axon segments are at or near the axon tip; bar, 20 μm. (B) Quantification of axon:dendrite polarity index for surface NgCAM or TfR coexpressed with Stx constructs. NgCAM polarity index was decreased in neurons coexpressing Stx3Δ38 or Stx3_AAA, whereas TfR, a somatodendritic cargo, was somatodendritic under all conditions tested (see images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163671#pone.0163671.s004" target="_blank">S4 Fig</a>). Error bars, SEM; N = 20–21 for NgCAM, 13–14 for TfR; * P ≤ 0.0001. (C) Nrxn is targeted to the axon when expressed alone (with tdTomato), but is mislocalized to the somatodendritic region when coexpressed with Stx3 mutants. Bar, 20 μm. (D) Axon:dendrite polarity index for surface Nrxn was reduced when Nrxn was coexpressed with Stx3Δ38 or Stx3_AAA compared to control. Error bars, SEM; N = 26–30; * P<0.0001.</p