Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

We thank Carl-Henrik Heldin (Uppsala University, Sweden) for his generous gift of the PS1 pMad antibody, Hugo Bellen, Corey Goodman, Janis Fischer, Graeme Davis, Guillermo Marques, Michael O'Connor, Kate O'Connor-Giles, and the Bloomington Drosophila Stock Center for flies strains, the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies to Wit and CSP; Marie Phillips for advice on membrane fractionation; Avital Rodal, Kate O'Connor-Giles, Ela Serpe, Kristi Wharton, Mojgan Padash-Barmchi for discussions or comments on the manuscript. We also thank Jody Summers at OUHSC for her generosity in letting us to use her confocal microscope.Conceived and designed the experiments: PAV TRF LRC BZ. Performed the experiments: PAV TRF LRC SMR HB NER BZ. Analyzed the data: PAV TRF LRC SMR HB NER BZ. Wrote the paper: PAV TRF BZ.Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.Yeshttp://www.plosone.org/static/editorial#pee

Interleukin 6 Function in the Skin and Isolated Keratinocytes Is Modulated by Hyperglycemia

Diabetes currently affects over twenty-five million Americans. Annual health care cost of diabetes exceeds $254 billion and is associated with a distinct set of diabetic complications that include delayed wound healing and diabetic ulcers. Interleukin 6 (IL-6) plays an important role in wound healing and is known to be elevated in the serum of both type I and type II diabetes patients. This study assesses the expression and function of IL-6 in the hyperglycemic epidermis and keratinocyte culture. Streptozotocin-treated mice were wounded six weeks after induction of hyperglycemia. Wound closure, protein, and mRNA expression were assessed up to 13 days of postwounding. Wound closure was delayed 4-5 days in hyperglycemic animals. Hyperglycemic wounds displayed greater IL-6 and IL-6Rα protein expression at 1, 7, and 10 days of postwounding compared to euglycemic control. However, IL-6Rα mRNA expression was reduced at all time points beyond day 1, while IL-6 mRNA expression did not significantly differ at any time point. SOCS3 mRNA expression was higher in the hyperglycemic skin at every time point. Imaging of fluorescent immunohistology also revealed significantly lower expression of SOCS3, but higher nuclear pSTAT3 in the epidermis of the hyperglycemic skin. Primary mouse keratinocytes cultured in high glucose for 7 days displayed 2-fold higher IL-6Rα mRNA and higher rmIL-6-induced nuclear pSTAT3, but lower SOCS3 basal levels compared to normal glucose-cultured cells. Thus, it appears that delayed diabetic skin wound healing is associated with increased induction and expression of IL-6 and its receptor, but its function in epidermal keratinocytes may be impaired

Novel pyrazolo[4,3-d]pyrimidine microtubule targeting agents (MTAs): Synthesis, structure–activity relationship, in vitro and in vivo evaluation as antitumor agents

The design, synthesis, and biological evaluation of a series novel N1‑methyl pyrazolo[4,3-d]pyrimidines as inhibitors of tubulin polymerization and colchicine binding were described here. Synthesis of target compounds involved alkylation of the pyrazolo scaffold, which afforded two regioisomers. These were separated, characterized and identified with 1H NMR and NOESY spectroscopy. All compounds, except 10, inhibited [3H]colchicine binding to tubulin, and the potent inhibition was similar to that obtained with CA-4. Compounds 9 and 11–13 strongly inhibited the polymerization of tubulin, with IC50 values of 0.45, 0.42, 0.49 and 0.42 μM, respectively. Compounds 14–16 inhibited the polymerization of tubulin with IC50s near ∼1 μM. Compounds 9, 12, 13 and 16 inhibited MCF-7 breast cancer cell lines and circumvented βIII-tubulin mediated cancer cell resistance to taxanes and other MTAs, and compounds 9–17 circumvented Pgp-mediated drug resistance. In the standard NCI testing protocol, compound 9 exhibited excellent potency with low to sub nanomolar GI50 values (≤10 nM) against most tumor cell lines, including several multidrug resistant phenotypes. Compound 9 was significantly (P \u3c 0.0001) better than paclitaxel at reducing MCF-7 TUBB3 (βIII-tubulin overexpressing) tumors in a mouse xenograft model. Collectively, these studies support the further preclinical development of the pyrazolo[4,3-d]pyrimidine scaffold as a new generation of tubulin inhibitors and 9 as an anticancer agent with advantages over paclitaxel

The 3-D conformational shape of N-naphthyl-cyclopenta[d]pyrimidines affects their potency as microtubule targeting agents and their antitumor activity

A series of methoxy naphthyl substituted cyclopenta[d]pyrimidine compounds, 4-10, were designed and synthesized to study the influence of the 3-D conformation on microtubule depolymerizing and antiproliferative activities. NOESY studies with the N,2-dimethyl-N-(6\u27-methoxynaphthyl-1\u27-amino)-cyclopenta[d]pyrimidin-4-amine (4) showed hindered rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. In contrast, NOESY studies with N,2-dimethyl-N-(5\u27-methoxynaphthyl-2\u27-amino)-cyclopenta[d]pyrimidin-4-amine (5) showed free rotation of the naphthyl ring around the cyclopenta[d]pyrimidine scaffold. The rotational flexibility and conformational dissimilarity between 4 and 5 led to a significant difference in biological activities. Compound 4 is inactive while 5 is the most potent in this series with potent microtubule depolymerizing effects and low nanomolar IC values in vitro against a variety of cancer cell lines. The ability of 5 to inhibit tumor growth in vivo was investigated in a U251 glioma xenograft model. The results show that 5 had better antitumor effects than the positive control temozolomide and have identified 5 as a potential preclinical candidate for further studies. The influence of conformation on the microtubule depolymerizing and antitumor activity forms the basis for the development of conformation-activity relationships for the cyclopenta[d]pyrimidine class of microtubule targeting agents

pMad fails to accumulate in the nucleus of motoneurons in <i>lqf</i> mutants.

<p>(A–E) Representative images of motoneuron nuclei in <i>Drosophila</i> 3^rd instar larval ventral nerve cord (VNC). pMad signal alone is shown on the left, whereas neuronal membrane (marked by HRP, green) and pMad (red) are shown on the right. (B) In <i>lqf</i> mutants, nuclear pMad is significantly reduced relative to control (CS, A), whereas neuronal overexpression of Lqf results in an increase in nuclear localized pMad (C). Similarly, two other endocytotic mutants <i>endo</i> (D) and <i>nwk</i> (E) have increased levels of nuclear pMad. (F) Quantification of nuclear pMad levels in A–E. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (G–H') Dissected and fixed 3^rd Instar larvae were stained with anti-CSP, and segmental nerves (which contain hundreds axons of sensory and motoneurons) were examined for general traffic defects, which would be apparent as CSP accumulations. There are no significant CSP accumulations in either the wild type or <i>lqf</i> mutants. Scale bars are 10 µm in all images. n values denote number of nuclei for each genotype, quantified from three animals per genotype. Control (CS), <i>nwk</i> (<i>nwk¹/nwk¹</i>), <i>endo</i> (<i>endo^A/endo^Δ4</i>), <i>lqf</i> (<i>lqf^ARI/lqf^FDD9</i>), Lqf^O/E (Elav^C155-Gal4/+; UAS-Lqf/+).</p

Lqf is required for synaptic overgrowth and pMad retrograde transport in <i>spinster</i> mutants.

<p>(A–F) Representative images of bouton morphology and pMad levels at the NMJ (A, C, E) and motoneuron nuclei (B, D, F) in <i>Drosophila</i> 3^rd instar larval NMJs from control larvae (CS, A–B), <i>spin</i> mutants (C–D) and <i>spin;lqf</i> double mutants (E–F). Synaptic boutons are overgrown in <i>spin</i> mutants (C), and this overgrowth is suppressed in the <i>spin;lqf</i> double mutants (E). (G) Quantification of synaptic bouton number in <i>spin</i> and <i>spin;lqf</i> mutants. Taken from three different animals for each genotype, n values represent the number of NMJs quantified. (H) Quantification of pMad intensity in boutons (white bars) and motoneuron nuclei (black bars). n = 5 NMJs from three larvae and 20 nuclei from five different larvae. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. Control (CS), <i>spin</i> (<i>spin⁴/spin⁵</i>), <i>spin, lqf</i> (<i>spin⁴/spin⁵; lqf^ARI/lqf Df</i>).</p

Lqf interacts with Wishful thinking (Wit) to regulate synapse growth.

<p>(A–G) Representative images of synaptic bouton morphology in <i>Drosophila</i> 3^rd instar larval NMJs from the indicated genotypes. Compared to control boutons (A), both <i>lqf</i> (<i>lqf^ARI</i>/<i>lqf</i>^FDD9) and <i>wit</i> mutants (<i>wit</i>^B11/<i>wit</i>^A12) (B and C, respectively), have fewer boutons, as does the <i>wit, lqf</i> double mutant (<i>wit^A12</i>/<i>wit^B11</i>; <i>lqf^ARI</i>/<i>lqf</i> Df; D). Neuronal overexpression of Lqf induces synaptic growth with increased number of branches and small satellite boutons and the presence of abnormally large ‘growth cone’-like boutons (Elav^C155-Gal4/+; UAS-Lqf/+; E), which is suppressed partially by removal of a single copy of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/+</i>; F), or suppressed completely by removal of both copies of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/wit^B11</i>; G). H). Quantification of A–G. Error bars represent SEM, n values represent the number of animals per genotype. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (I) Co-immunoprecipitation of Flag-tagged Lqf from adult brain lysates from either control flies (containing no Flag), or flies expressing Flag-tagged Lqf, shows Wit co-immunoprecipitates with Lqf. Abbreviations are as follows: Immunoprecipitation (IP); Immunoblot (IB); Input (I); Supernatant (S); Immunoprecipitate (IP).</p