Munc13 controls the location and efficiency of dense-core vesicle release in neurons

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically

SNAP-25 gene family members differentially support secretory vesicle fusion

Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that Snap-25-null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days in vitro (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively

Conservation of SUMOplot predicted tomosyn SUMOylation sites.

<p>Protein alignment of tomosyn isoforms in different species, as well as the homologous proteins Lgl and yeast Sro7 and Sro77. Lysine residues that are predicted to be subject to SUMOylation in mouse tomosyn are highlighted with black boxes. SUMOplot prediction scores for (A) tomosyn-m2 K279, (B) tomosyn-m1 K285, (C) tomosyn-m1 K298 / tomosyn-m2 K309 and (D) tomosyn m-1 K730 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091697#pone-0091697-t001" target="_blank">table 1</a>. Colours represent amino acid physicochemical properties: small (red), acidic (blue), basic (magenta), hydroxyl/sulfhydryl/amine/G (green).</p

SUMO-2/3 modification of tomosyn-1.

<p>(A) Schematic representation of the tomosyn-m1 construct used in the immunoprecipitation experiment. Light grey: β-propeller domains, tomosyn dark grey: synaptobrevin-like coiled coil domain. Tomosyn phosphorylation (P) and SUMOylation (S) sites are also depicted. A small myc-tag is depicted at the tomosyn C-terminus. Immunoprecipitation of mock- and tomosyn-1-myc transfected HEK293T cell lysates with an anti-myc antibody was analyzed by immunoblotting with (B) anti-tomosyn, (C) anti-SUMO-1 or (D) anti-SUMO-2/3 antibodies. A fraction (2.5%) of the total cell lysate was loaded on the gel to verify protein expression (‘input’), the rest of the sample was used for immunoprecipitation (‘anti-myc IP’). The SUMO-2/3 antibody detected a band the size of tomosyn-1-myc (*; 126 kDa), indicating SUMOylation of tomosyn by SUMO-2/3.</p

NEM dependent PIASγ / tomosyn-1 interaction.

<p>(A) Schematic representation of the constructs used in immunoprecipitation experiments. Tomosyn light grey: β-propeller domains, tomosyn dark grey: synaptobrevin-like coiled coil domain. Tomosyn phosphorylation (P) and SUMOylation (S) sites are also depicted. A small myc-tag is depicted at the tomosyn C-terminus. PIASγ light grey: DNA-binding SAP-domain, dark grey: MIZ-type zinc finger domain. The N-terminal FLAG-tag is also depicted. (B) FLAG-PIASγ co-precipitates with full-length tomosyn-1-myc in the presence of N-ethylmaleimide (NEM). Lysate from HEK293T cells co-transfected with tomosyn-1-myc (126 kDa) and FLAG-PIASγ (57 kDa, but reported to run higher on a Western blot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091697#pone.0091697-Ihara1" target="_blank">[56]</a>) was subjected to immunoprecipitation using an anti-myc antibody with/without NEM in the lysis buffer. Antibody was omitted in the negative control. In the positive control, syntaxin (35 kDa) was expressed instead of FLAG-PIASγ. A fraction (2.5%) of the total cell lysate was loaded on the gel to verify protein expression (‘input’), the rest of the sample was used for immunoprecipitation (‘anti-myc IP’). (C) Tomosyn-1-myc co-precipitated in a reverse immunoprecipitation with an anti-FLAG antibody pulling down FLAG-PIASγ in the presence of NEM.</p

Yeast two-hybrid interaction of tomosyn-1 and PIASγ.

<p>(A) Tomosyn amino acids 540-1116 (Cter) or 1028-1116 (CoiledCoil) were used as bait in a yeast two-hybrid (Y2H) experiment (light grey: β-propeller domains, dark grey: synaptobrevin-like coiled coil domain, phosphorylation (P) and SUMOylation (S) sites are also depicted), next to an empty bait vector pBD-GAL4, in combination with prey constructs expressing (B) PIASγ (light grey: DNA-binding SAP-domain, dark grey: MIZ-type zinc finger domain) or syntaxin sequences, next to an empty prey vector pAct2. Yeast colonies were incubated on (C) histidine containing medium (no selection), or medium (D) lacking histidine (medium stringent selection) or (E) lacking histidine and adenine (stringent selection). Stringent selection indicated a strong PIASγ interaction with the larger Cter fragment of tomosyn-1 as well as syntaxin binding to tomosyn-1 CoiledCoil fragment.</p

A Monoclonal Antibody TrkB Receptor Agonist as a Potential Therapeutic for Huntington’s Disease

<div><p>Huntington’s disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for <i>in vivo</i> proof of concept studies in transgenic HD models.</p></div

TrkB signaling and assay cascades.

<p>(A) TrkB signalling cascade showing proximal and distal assay measurement points. Red arrows represent BDNF-induced trans-phosphorylation events within the intracellular tyrosine kinase domains. Changes in TrkB phosphorylation/activation detected by (1) Invitrogen CellSensor®; (2) DiscoveRx PathHunter®; and (3) MSD® pAKT assays. (B) Screening cascade used to characterize TrkB modulators.</p

Functional activity of digested 38B8 mAb in the TrkB NFAT reporter gene assays.

<p>CellSensor® TrkB-NFAT-bla CHO-K1 cells were stimulated with (A) BDNF, mAb 38B8, 38B8 Fab or 38B8 F(ab’)2 over the indicated concentration range for 5 hours (agonist mode) or (B) 38B8 Fab for 1 hour prior to 0.3 nM BDNF stimulation for 4 hours (antagonist mode) before beta-lactamase assay was performed as described in Methods. % of control (maximal BDNF concentration  =  9 nM) values were plotted for the indicated concentrations of each ligand (n = 2 ± SD for each data point).</p

TrkB antibody panel and associated activities.

#<p>Reactivity as described by suppliers.</p