Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ40 and Aβ42, the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

<div><p>Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 <i>versus</i> Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.</p></div

Additional file 1: Figure S1. of Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo

A representative section from the lumbar part of the spinal cord. The regions under the blue-lined rectangular boxes show the areas where CD4+ T cells and SMI32-positive axons were quantified and analyzed. (PPTX 6495 kb

mTORC2 inactivation inhibits focal adhesion kinase activity.

<p>HUVECs were transfected with siRict¹ or siAkt1, then stimulated with 20 ng/mL VEGF for 10 minutes as indicated. <b>A</b>) A representative Western blot of EC phospho-focal adhesion kinase (P-FAK), total FAK, total Akt1, total rictor and actin. <b>B)</b> Quantitation of P-FAK (n = 4 independent experiments, *<i>P</i><0.05 by ANOVA). <b>C)</b> A representative Western blot of EC phospho-eNOS, and phospho-Src, illustrates that mTORC2 disruption, but not Akt1 inactivation, blocks VEGF-stimulated Src activation (n = 3 independent experiments). Knockdown of either rictor or Akt1 similarly blunts eNOS phosphorylation. <b>D)</b> Quantitation of P-Src (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). <b>E)</b> The effect of sustained mTORC1 or mTORC1/2 inhibition on EC FAK activation. HUVECs were treated with PP242 or rapamycin and stimulated with VEGF overnight. A representative Western blot of EC P-FAK, and P-S6K (n = 3 independent experiments). <b>F)</b> Quantitation of P-FAK (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p

An activation-independent role of transcription factors in insulator function

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains

Sustained mTORC1, but not mTORC2, inhibition activates Akt.

<p>HUVECs were treated by rapamycin, two different small interfering RNAs against rictor (siRict¹ and siRict²) or raptor (siRapt), or non-silencing siRNA (siNS), then stimulated with 20 ng/mL VEGF as indicated. Akt phosphorylation and S6K phosphorylation were evaluated as described in Materials and Methods. <b>A</b>) A representative Western blot of the effect of Rapamycin pretreatment for 1 hour on VEGF-stimulated Akt and S6 kinase phosphorylation in HUVECs. <b>B)</b> Quantitation of phospho-S6K. <b>C)</b> Quantitation of phospho-Akt (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA). The effect of rapamycin treatment of HUVEC for 24 hours. <b>D)</b> A representative Western blot of HUVEC phospho-Akt, total-Akt1 and tubulin over a range of concentration of rapamycin exposure. <b>E)</b> Quantitation of phospho-Akt (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA). The effect of mTORC1 <i>versus</i> mTORC2 disruption on Akt signaling in EC. <b>F)</b> A representative Western blot of HUVEC rictor, raptor, phospho-Forkhead box protein O1/3 (P-FOXO1/3), phospho-Akt, total Akt1, phospho-S6K, total S6K, and actin after treatment with siRapt, siRict or siNS. <b>G)</b> Quantitation of phospho-Akt. <b>H)</b> Quantitation of phospho-S6K (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA).</p