Development of Potent Fluorescent Polyamine Toxins and Application in Labeling of Ionotropic Glutamate Receptors in Hippocampal Neurons

The natural product argiotoxin-636 (ArgTX-636) found in the venom of the Argiope lobata spider is a potent open-channel blocker of ionotropic glutamate (iGlu) receptors, and recently, two analogues, ArgTX-75 and ArgTX-48, were identified with increased potency and selectivity for iGlu receptor subtypes. Here, we have exploited these analogues as templates in the development of fluorescent iGlu receptor ligands to be employed as unique tools for dynamic studies. Eighteen fluorescent analogues were designed and synthesized, and subsequently pharmacologically evaluated at three iGlu receptor subtypes, which resulted in the discovery of highly potent fluorescent iGlu receptor antagonists with IC₅₀ values as low as 11 nM. The most promising ligands were further characterized showing retention of their mechanism of action, as open-channel blockers of iGlu receptors, as well as preservation of the photophysical properties of the incorporated fluorophores. Finally, we demonstrate the applicability of the developed probes for imaging of iGlu receptors in hippocampal neurons

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

<div><p>Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient <i>Drosophila</i> and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the <i>Drosophila</i> brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a <i>Drosophila</i> model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.</p></div

PICK1 and ICA69 co-localize in the Golgi compartment of COS7 cells and promote punctate distribution of GFP-tagged chromogranin A (CgA-GFP) in the cytosol.

<p>(A) Confocal images of COS-7 cells transfected with GFP-PICK1 (top), HA-ICA69 (middle), or GFP-PICK1 and HA-ICA69 (bottom). From left, the channels show GFP-PICK1 in green, HA-ICA69 in red, and endogenous giantin in blue. Upon co-expression, GFP-PICK1 and ICA69 co-localize with giantin in the Golgi compartment. Scale bar, 10 µm. (B) Confocal images of COS-7 cells transfected with CgA-GFP and pcDNA3 (top) or together with mycPICK1 and HA-ICA69 (bottom). From left, the green channel shows CgA-GFP, the red channel shows mycPICK1, and the blue channel shows HA-ICA69. Insets illustrate the marked increase in punctuate CgA structures in cells co-transfected with mycPICK1 and HA-ICA69. Outlines of the cells are shown in the green channel in white. Scale bar, 10 µm. Images are representative of <i>n</i> = 50 (CgA-GFP) and <i>n</i> = 44 (CgA-GFP+mycPICK1+HA-ICA69) cells from three independent sessions. (C) Quantification of the punctuate CgA-GFP signal relative to the total CgA-GFP signal as a measure of the efficiency of Golgi exit. ***<i>p</i><0.001 in Student's <i>t</i> test.</p

PICK1 is localized to vesicles exiting the Golgi in cultured GH1 cells, and knockdown of PICK1 reduces GH immunosignal in GH1 cells.

<p>(A) PICK1 is expressed in GH-producing GH1 cells, and PICK1 knockdown reduces GH content. Confocal images of GH1 cells immunostained for PICK1 (blue) and GH (red). Cells transfected with shRNA against PICK1 are identified by obligate coexpression of green fluorescent protein (EmGFP) (green) and are outlined. Insets (squares) show area with (i) no colocalization of PICK1 and GH and (ii) partial colocalization of PICK and GH. Scale bar, 10 µm. (B and C) Quantification of PICK1 immunosignal (B) and GH immunosignal (C) in GFP-positive cells transfected with either of two different shRNAs against PICK1 (52 and 53). Data are % of signal in surrounding non-transfected cells (mean ± SE) for control shRNA (shControl) (<i>n</i> = 48), shPICK1 (52) (<i>n</i> = 69), and shPICK1 (53) (<i>n</i> = 75). ***<i>p</i><0.001, compared to shControl, Mann–Whitney rank sum test. (D) Quantification of the PICK1 co-localization with GH using Van Steensel's cross-correlation function, which reports the Pearson cross-correlation as a function of the relative movement of the two channels with respect to each other. The low but sharp peak close to Δx = 0 indicates partial but specific co-localization. (E) Co-localization of PICK1 with Giantin, TGN38, and syntaxin 6. Confocal laser scanning micrographs of GH1 cells immunostained for endogenous PICK1 (red) and the cis-Golgi marker giantin (top), the trans-Golgi (TGN) marker TGN38 (middle), and the TGN/immature vesicle marker syntaxin 6 (bottom) (all in green). Insets highlight area where the PICK1 signal was either enclosed by, or closely associated with, the Golgi markers (giantin, top, and TGN38, middle) or partially co-localizing (syntaxin 6, bottom). (F) Quantification of PICK1 colocalization with giantin, TGN38, and syntaxin 6 using Van Steensel's cross-correlation function. The broad peak for giantin is characteristic for large adjacent structures, whereas the high and sharp peak close to Δx = 0 for syntaxin 6 indicates specific co-localization. TGN38 is intermediate between these distributions, suggesting a partial overlap of the structure with PICK1. Co-localization was quantified for 10–20 cells from three independent experiments, and data are means ± SE. (G) Dashed box (left) from (E, Bottom) with magnification and gain adjusted for visualization of the punctate staining of PICK1 (red) and syntaxin 6 (green). Intensity profile (right) through several punctae (white line in lower, merged picture on the left) shows peaks corresponding to punctae for both PICK1 and syntaxin 6. All immunocytochemistry data were obtained from at least three independent experiments, and the cells are representative of multiple cells imaged in each experiment.</p

Decreased GH storage and secretion in PICK1-deficient mice.

<p>(A) The livers of PICK-1-deficient mice had a significantly lower weight than those of WT mice (**<i>p</i><0.01). (B) PICK1-deficient mice exhibited a decreased level of plasma IGF-1 (*<i>p</i><0.05). (C) Decreased relative expression level of IGF-1 mRNA in the liver of PICK1-deficient mice versus WT mice (**<i>p</i> = 0.0045). Measurements in (A), (B), and (C) were performed in samples from 35–38-wk-old WT (+/+) or PICK1-deficient mice (−/−) after overnight fast. Data are means ± SE (<i>n</i> = 10–14). (D) PICK1-deficient mice display reduced femoral length. Measurements were done on 39–41-wk-old WT (+/+) and PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 4–5). *<i>p</i><0.05, compared to WT. (E) PICK1-deficient mice display decreased ghrelin-induced GH secretion. Basal plasma GH level and the level 5 min after ghrelin administration were determined in anesthetized WT (+/+) and PICK1-deficient mice (−/−) at the age of 7–9 wk. White bars, WT (+/+); black bars, PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 7–9). *<i>p</i><0.05, compared to WT. (F) PICK1-deficient mice display decreased GH content in the pituitary. Pituitaries were taken from 34-wk-old WT (+/+) or PICK1-deficient mice (−/−). Data are means ± SE (<i>n</i> = 4). **<i>p</i><0.01. (G) Relative expression levels of GH and GHR mRNA in pituitaries, determined by RT-PCR analysis (35–38-wk-old mice). White bars, PICK1 +/+: black bars, PICK1 −/−. Data are means ± SE (<i>n</i> = 7–8). *<i>p</i><0.05.</p

Interdependent expression of PICK1 and ICA69 in <i>Drosophila</i>.

<p>(A) Immunostainings for ICA69 and PICK1 in a single peptidergic neuron in the protocerebrum of adult <i>Drosophila</i> brain. HA-tagged ICA69 targeted to peptidergic cells (<i>c929-GAL4/+; UAS-ICA69-HA/+</i>) was detected in parallel with endogenous PICK1. Arrows indicate examples of PICK1/ICA69 co-localization. Scale bar, 10 µm. (B and C) <i>Drosophila</i> ICA69-HA co-immunoprecipitates PICK1 from fly head extracts. Flies were co-expressing ICA69-HA and PICK1 under the pan-neuronal elav-GAL4 driver (<i>elav-GAL4/+; UAS-ICA69-HA/UAS-PICK1A</i>). (B) Immunoprecipitation (IP) with anti-HA antibody and immunoblotting (IB) with anti-PICK1 antibody. Left lane, control without antibody; right lane, lysate. (C) Immunoprecipitation (IP) with rat anti-HA antibody (3F10) and immunoblotting (IB) with mouse anti-HA (16B12) antibody. Left lane, control without antibody; right lane, lysate. (D) Dependence of ICA69 expression on PICK1 expression in <i>Drosophila</i>. ICA69-HA immunoreactivity is shown in control brain (<i>c929-GAL4</i>, <i>PICK¹/+; UAS-ICA69-HA/+</i>) and in PICK1-null mutant brain (<i>c929-GAL4, PICK¹/PICK²; UAS-ICA69-HA/+</i>). pi, pars intercerebralis; pc, protocerebrum; ol, optic lobe. Pictures are representative of six fly brains stained in three experiments. (E) Dependence of PICK1 expression on ICA69 expression in <i>Drosophila</i>. PICK1 immunosignal in control brain without RNAi transgene (<i>ELAV-GAL4/UAS-DCR2;;TM3/+</i>) and in brain with pan-neuronal expression of ICA69 hairpin RNA (<i>ELAV-GAL4/UAS-DCR2;;UAS-ICA-RNAi/+</i>) (ICA69-RNAi). Scale bar, 100 µm. Pictures are representative of six fly brains stained in three experiments. (F) Western blotting of head extracts from control and ICA69 RNAi flies. (G) Quantification of the data in (F). Protein levels are normalized to ELAV. Data are means ± SE, <i>n</i> = 6, <i>p</i><0.05. (H and I) Quantification by RT-PCR of ICA69 mRNA (H) and PICK1 mRNA (I) levels in heads from <i>ICA69</i> knockdown (ICA69-RNAi), <i>PICK1¹</i>, and control flies. Data are means ± SE, <i>n</i> = 6 for control and ICA69-RNAi, <i>n</i> = 4 for <i>PICK1¹</i>, *<i>p</i><0.05, **<i>p</i><0.01.</p

PICK1 associates transiently with the Golgi compartment and is capable of tubular deformation of liposomes in vitro.

<p>(A) Brief (5 min) brefeldin A treatment traps PICK1 in the trans-Golgi. Confocal images of GH1 cells immunostained for PICK1, and the trans-Golgi marker TGN38, and the trans-Golgi/immature vesicle marker syntaxin 6 before (top) and after 5 min BFA treatment (bottom). Panels show from left signal from immunolabeled PICK1 (Alexa Fluor 568 signal), signal from TGN38 (Alexa Fluor 488 signal), signal from syntaxin 6 (Alexa Fluor 647 signal), and overlay of the three channels. Insets highlight an area with overlapping localization of PICK1 and syntaxin 6 but with PICK1 adjacent to TGN38 (top) and colocalization of PICK1 with both syntaxin and TGN38 (bottom). (B) Quantification of the PICK1 colocalization with TGN38 and syntaxin 6 after 5 min BFA treatment using Van Steensel's cross-correlation function, which reports the Pearson cross-correlation as a function of the relative movement of the two channels with respect to each other. Both syntaxin 6 and TGN38 show a sharp peak of similar height close to Δx = 0, indicating specific co-localization. Co-localization was quantified for 10–20 cells from three independent experiments, and data are means ± SE. Note that the drop in peak values compared to no BFA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001542#pbio-1001542-g006" target="_blank">Figure 6F</a>) likely reflects the increased diffuse localization of both markers after BFA treatment. (C) Fast time-lapse dual color live confocal imaging showing transient association of YFP-PICK1 BAR with the Golgi marker GalT-Cerulean. Images are maximal intensity projections of representative time-lapse series for YFP-PICK1 BAR (left), YFP PICK1 BAR V121E-L125E (middle), and YFP-PICK1 BAR 3KE (right). The gray scale projections (left images) show the YFP channel alone, indicating highly dynamic behavior of YFP PICK1 BAR (left) and more static behaviour of YFP-PICK1 BAR V121E-L125E (middle) punctae, whereas no punctae are seen for YFP-PICK1 BAR 3KE (right). The dual color projections (right images, YFP channel in yellow, Cerulean channel in blue) show that most of the activity for YFP-PICK BAR (left) is lining the Golgi, whereas YFP-PICK1 BAR V121E-L125E (middle) shows activity throughout the cell. Circles indicate transiently appearing punctate structures (lasting for less than 10 frames, ∼30 s). Crosses indicate stable punctate structures lasting for more than 10 frames. (D) Time-lapse series of two transient punctate structures of YFP-PICK1 BAR, and (E) two stable punctate structures of YFP-PICK1 BAR V121E-L125E. Scale bar, large images 5 µm, time lapse 1 µm. (F) Tubulation of surface-immobilized artificial giant membrane vesicles by the PICK1 BAR domain. Purified and Alexa Fluor 488–labeled GST-PICK1 BAR or GST-PICK1 BAR V121E-L125E (control) with impaired membrane binding capacity. Protein was incubated at room temperature with surface immobilized and fluorescently labeled vesicles before confocal fluorescent imaging. No effect was seen for the mutant, whereas massive tubulation was seen with GST-PICK1 BAR. Arrow indicates a budding vesicle from a single GV.</p

Rescue of the phenotype of PICK1-deficient mice by GH administration.

<p>(A) Lean body mass was evaluated by MRI scanning before GH administration and subsequently once every week both in PICK1-deficient mice (black squares) and in saline-treated WT littermates (white circles). Before treatment the difference between the two groups was highly significant (**<i>p</i> = 0.0027), after a week the difference decreased but was still significant (*<i>p</i> = 0.050), and after 2 and 3 wk no significant difference were observed. (B) IGF-1 mRNA level in the liver as determined by RT-PCR was significantly lower in PICK1-deficient mice compared to untreated mice (*<i>p</i> = 0.0045), however this difference was not observed after GH-treatment for 3 wk. (C) OGTT and ITT on GH-treated PICK1-deficient mice (black circles) and the saline-treated WT littermate controls (white circles). Significant difference was observed in the OGTT, whereas no difference was observed in ITT. Data are expressed as mean ± SE and analyzed by two-way ANOVA in (A) and Student's <i>t</i> test in (B–D) (<i>n</i> = 4–8 in all the experiments). All experiments have been reproduced in another cohort of mice (<i>n</i> = 3–4), where the age of the mice was 4 wk higher.</p

Dysregulation of glucose homeostasis in PICK1-deficient mice.

<p>(A) Impaired glucose tolerance in PICK1-deficient mice. OGTT was done in 10–13-wk-old WT (+/+) and PICK1-deficient mice (−/−) after overnight fast. White circles, PICK1 +/+; black circles, PICK1 −/−. The inset shows area under curve (AUC). Data are means ± SE (<i>n</i> = 9). **<i>p</i><0.01. (B) Mice deficient in PICK1 display decreased insulin secretion during OGTT. White bars, PICK1 +/+: black bars, PICK1 −/−. Data are means ± SE. *<i>p</i><0.05. (C) Mice deficient in PICK1 (27–29 wk) display preserved or even improved insulin sensitivity in the ITT. White circles, PICK1 +/+; black circles, PICK1 −/−. Data are means ± SE (<i>n</i> = 9–11). AUC was significantly lower for the PICK1-deficient mice compared to WT mice. *<i>p</i><0.05. (D) The protein expression level of the insulin receptor as determined by quantification of Western blots on liver samples from 35–38-wk-old mice after overnight fasting was significantly increased in PICK1-deficient mice (−/−) compared to WT (+/+) mice (means ± SE, *<i>p</i> = 0.024). Western blots with insulin receptor and actin immunoreactivity of two representative samples out of 13 PICK−/− and 10 PICK+/+ samples are shown below. (E) The plasma levels of triglycerides (TG) after overnight fasting were significantly decreased in PICK1-deficient mice (−/−) compared to WT (+/+) mice (154±15 versus 103±9, <i>n</i> = 10–13, **<i>p</i> = 0.0056).</p

PICK1 deficiency leads to loss and altered localization of DCVs in the pituitary.

<p>(A) PICK1 is expressed in GH-producing cells of the pituitary. Confocal images of 15 µm slices from the pituitary of WT (+/+) (top) and PICK1-deficient mice (−/−) (bottom), immunostained for PICK1 (green) and GH (red). Insets show cells with characteristic intense arc-shaped distribution of GH and partial overlap with PICK1. Scale bar, 10 µm. Pictures are representative of stainings of seven pituitaries from WT and PICK1-deficient mice. (B) The number of DCVs is reduced in pituitary cells from PICK1-deficient mice. Transmission electron micrographs of uranyl acetate and lead citrate-stained ultrathin slices from the pituitary of WT (+/+) (top) and PICK1-deficient mice (−/−) (bottom). Scale bars, left, 20 µm. Pictures are representative of several pictures from five WT (+/+) and four PICK1-deficient (−/−) mice. (C) Quantification of the number of DCVs in 26 cells from WT (+/+) and 29 cells from PICK1-deficient (−/−) mice. **Average significantly different from control, <i>p</i> = 0.0022 in Student's <i>t</i> test. (D) Size distribution of DCVs in GH cells of the pituitary is unaltered in PICK1-deficient mice. The data show the relative frequency of different sizes of DCV in cells from PICK1-deficient mice (black) (<i>n</i> = 3,798) and in cells from WT mice (white) (<i>n</i> = 7,021).</p