Glucagon-Like Peptide-1 Gene Therapy

Glucagon-like peptide 1 (GLP-1) is a small peptide component of the prohormone, proglucagon, that is produced in the gut. Exendin-4, a GLP-1 receptor agonist originally isolated from the saliva of H. suspectum or Gila monster, is a peptide that shares sequence and functional homology with GLP-1. Both peptides have been demonstrated to stimulate insulin secretion, inhibit glucagon secretion, promote satiety and slow gastric emptying. As such, GLP-1 and Exendin-4 have become attractive pharmaceutical targets as an adjunctive therapy for individuals with type II diabetes mellitus, with several products currently available clinically. Herein we summarize the cell biology leading to GLP-1 production and secretion from intestinal L-cells and the endocrine functions of this peptide and Exendin-4 in humans. Additionally, gene therapeutic applications of GLP-1 and Exendin-4 are discussed with a focus on recent work using the salivary gland as a gene therapy target organ for the treatment of diabetes mellitus

Carboxypeptidase E/NFα1: a new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways.

Mice lacking Carboxypeptidase E (CPE) exhibit degeneration of hippocampal neurons caused by stress at weaning while over-expression of CPE in hippocampal neurons protect them against hydrogen peroxide-induced cell death. Here we demonstrate that CPE acts as an extracellular trophic factor to protect neurons. Rat hippocampal neurons pretreated with purified CPE protected the cells against hydrogen peroxide-, staurosporine- and glutamate-induced cell death. This protection was observed even when hippocampal neurons were treated with an enzymatically inactive mutant CPE or with CPE in the presence of its inhibitor, GEMSA. Purified CPE added to the culture medium rescued CPE knock-out hippocampal neurons from cell death. Both ERK and AKT were phosphorylated within 15 min after CPE treatment of hippocampal neurons and, using specific inhibitors, both signaling pathways were shown to be required for the neuroprotective effect. The expression of the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), was up-regulated after hippocampal neurons were treated with CPE. Furthermore, hydrogen peroxide induced down-regulation of BCL-2 protein and subsequent activation of caspase-3 were inhibited by CPE treatment. Thus, this study has identified CPE as a new neurotrophic factor that can protect neurons against degeneration through the activation of ERK and AKT signaling pathways to up-regulate expression of BCL-2

Carboxypeptidase E Cytoplasmic Tail-Driven Vesicle Transport Is Key for Activity-Dependent Secretion of Peptide Hormones

Vesicular transport of peptide hormones from the cell body to the plasma membrane for activity-dependent secretion is important for endocrine function, but how it is achieved is unclear. Here we uncover a mechanism in which the cytoplasmic tail of transmembrane carboxypeptidase E (CPE) found in proopiomelanocotin (POMC)/ACTH vesicles interacts with microtubule-based motors to control transport of these vesicles to the release site in pituitary cells. Overexpression of the CPE tail in live cells significantly reduced the velocity and distance of POMC/ACTH- and CPE-containing vesicle movement into the cell processes. Biochemical studies showed that the CPE tail interacted with dynactin, which, in turn, recruited microtubule plus-end motors kinesin 2 and kinesin 3. Overexpression of the CPE tail inhibited the stimulated secretion of ACTH from AtT20 cells. Thus, the CPE cytoplasmic tail interaction with dynactin-kinesin 2/kinesin 3 plays an important role in the transport of POMC vesicles for activity-dependent secretion

Purified recombinant CPE protein protects against H₂O₂-induced neurotoxicity in primary cultured rat hippocampal neurons.

<p><b>A)</b> Bar graphs showing WST activity, indicative of cell viability, of hippocampal neurons treated with and without H₂O₂. Note the reduced cell viability after H₂O₂ treatment that was significantly attenuated by the pretreatment of the neurons with 0.4 µM and 1 µM purified CPE. <b>B)</b> Bar graphs showing LDH activity, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071578#pone-0071578-g001" target="_blank">figure 1</a>, in the culture media of hippocampal neurons treated with and without H₂O₂. Note that the H₂O₂-induced cytotoxicity was significantly attenuated by the pretreatment of the neurons with 0.4 µM and 1 µM purified CPE. <b>C)</b> Bar graphs showing WST activity of hippocampal neurons treated with purified CPE. Note that cell viability increased significantly by treatment with 0.4 µM and 1 µM CPE. <b>D)</b> Photomicrographs of hippocampal neurons with and without H₂O₂ treatment stained by TUNEL (green) and DAPI (blue). Note that the number of dead cells (green) increased significantly after H₂O₂ treatment (middle, left panel) and that pretreatment with 0.4 µM CPE protected the neurons (bottom, left panel). The bar graph represents the quantification of the dead cells as a % of the total number of cells determined by the DAPI staining. 500 cells were counted in each of 6 different dishes generated from embryos from 2 rats. <b>A–C</b>, one way ANOVA with Tukey post-hoc test, n = 5, *p<0.05 when compared to control cells; ^#p<0.05 compared to H₂O₂ treated only cells. <b>D</b>, Students t test, n = 6, ***p<0.001 compared to control cells; ^##p<0.01 compared to H₂O₂ treated only cells. Bar = 100 µm.</p

Neuroprotection by CPE involves BCL-2 and Caspase-3.

<p><b>A)</b> Bar graph showing the quantification by qRT-PCR of <i>Bcl-2</i> mRNA in primary cultured rat hippocampal neurons after treatment with 0.4 µM CPE for 3 h. Data is normalized against 18S RNA and presented as a % compared to untreated control (Ctrl) cells, (t-test, n = 3, *p<0.05). <b>B)</b> Representative Western blot analysis of BCL-2 protein in primary cultured hippocampal neurons, pretreated with 0.4 µM CPE for 24 h and subsequently challenged or not with H₂O₂ for 24 h. Actin was also analyzed and served as an internal control for protein load. <b>C)</b> Bar graphs showing the quantification of BCL-2 protein normalized to Actin and expressed as a % compared to untreated control (Ctrl) cells. Note that CPE significantly inhibited the H₂O₂-induced decrease in BCL-2 protein in primary cultured hippocampal neurons, (t-test, n = 4, ** p<0.01 compared to Ctrl; ^# p<0.05 compared to H₂O₂ treated only cells) <b>D)</b> Western blot analysis of cleaved caspase-3 in primary cultured hippocampal neurons pretreated with 0.4 µM CPE for 24 h and subsequently challenged or not with H₂O₂ for 24 h. Note that the H₂O₂-induced activation of caspase-3 is blocked by CPE. Western blot is representative of 2 independent experiments.</p

Purified recombinant CPE rescues cell death in CPE knock-out mouse hippocampal neurons.

<p><b>A)</b> Photomicrographs of 2 week cultures of hippocampal neurons derived from WT and CPE-KO embryos stained by TUNEL (green) and DAPI (blue). Note the higher number of dead cells (green) in the CPE-KO culture (KO) compared to the WT culture (WT). Culturing the CPE-KO neurons in the presence of 0.4 µM CPE significantly reduced the number of dead cells (KO+CPE). <b>B)</b> The bar graph represents the quantification of the dead cells as a % of the total number of cells determined by the DAPI staining. Students t test, n = 4, *p<0.05 compared to WT neurons; ^#p<0.05 compared to KO neurons not treated with CPE. Bar = 100 µm.</p

CPE containing conditioned medium protects against H₂O₂-induced cytotoxicity in primary cultured rat hippocampal neurons.

<p><b>A)</b> Western blot showing the presence of endogenous CPE in the cell lysate (CE) and conditioned medium (CM) of hippocampal neurons. No signal was obtained from the non-conditioned or fresh medium (FM). <b>B)</b> Western blot showing CPE protein in conditioned medium from primary hippocampal neurons transduced with adenovirus harboring LacZ and WT or E300Q CPE constructs. <b>C)</b> Bar graphs showing LDH activity, as an indicator of cellular cytotoxicity, in the culture media of hippocampal neurons treated with and without H₂O₂. Note the increased cytotoxicity after H₂O₂ treatment (LacZ/H₂O₂) compared to control cells (LacZ) that was significantly attenuated by the presence of either WT (CPE/H₂O₂) or E300Q (E300Q/H₂O₂) CPE. Students t test, n = 4, ***p<0.001 compared to LacZ control; ^###p<0.001, compared to LacZ/H₂O₂.</p

CPE activates ERK and AKT signaling pathways in primary cultured rat hippocampal neurons.

<p>Top panels: Representative Western blot analyses of hippocampal neuron lysates showing p-ERK 1/2 (<b>A</b>) and p-AKT (<b>B</b>) after 0, 15, 30 and 60 min treatment with 0.4 µM CPE. Total ERK (t-ERK 1/2) and total AKT (t-AKT) were also analyzed and served as internal controls. Bottom panels: Bar graphs showing the quantification of the p-ERK 1/2 (<b>A</b>) and p-AKT (<b>B</b>) signals normalized to t-ERK 1/2 and t-AKT, respectively. Note the increase in p-ERK and p-AKT after 15, 30 and 60 min treatment with CPE. Statistical analysis for <b>A</b> and <b>B</b> by one way ANOVA with Tukey post-hoc test, n = 4, *p<0.05 when compared to untreated control cells.</p

Mechanism of neuroprotection by CPE in primary cultured hippocampal neurons.

<p>CPE signals the MEK/ERK and PI3-K/AKT signaling pathways which up-regulate the expression of the anti-apoptotic protein, BCL-2, and inhibit the caspase cascade leading to a decrease in cleaved caspase-3, the apoptotic executioner.</p

Techniques in Neuropeptide Processing, Trafficking, and Secretion

Neuropeptides function as neurotransmitters and neuromodulators. They are synthesized as larger precursors at the rough endoplasmic reticulum (RER), trafficked to the trans-Golgi network (TGN), and sorted into granules of the regulated secretory pathway (RSP) for secretion in an activity-dependent manner. Polymorphisms found in human neuropeptide genes can lead to defects in trafficking and processing of the neuropeptide precursors, resulting in disease. Examples of mutations of human neuropeptide genes that have led to biosynthesis of precursors that were misrouted and only partially processed include insulin (1), and cocaine and amphetamine-regulated transcript (CART) peptide (2), giving rise to diabetes and obesity, respectively. A human valine to methionine mutation in the prodomain of brain-derived neurotrophic factor (BDNF) causes its inefficient sorting to the RSP and diminished activity-dependent secretion of BDNF from hippocampal neurons, resulting in memory deficits in these humans (3). With the sequencing of the human genome, increasing numbers of polymorphisms in neuropeptide genes will be identified. Studies on the trafficking, processing, and activity-dependent secretion of the mutant neuropeptide precursors will be useful in elucidating the molecular and cellular basis of diseases associated with the mutations. There are currently many paradigms and tools to study neuropeptide precursor trafficking, processing, and secretion, and these will be described in this chapter. These procedures are also applicable to studying the processing of other proteins such as neurotrophins.First Editio