Faculty Bibliography

Reports an error in "Epac2-dependent mobilization of intracellular Ca2+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in beta -cells of phospholipase C-epsilon knockout mice" by Igor Dzhura, Oleg G. Chepurny, Grant G. Kelley, Colin A. Leech, Michael W. Roe, Elvira Dzhura, Parisa Afshari, Sundeep Malik, Michael J. Rindler, Xin Xu, Youming Lu, Alan V. Smrcka and George G. Holz (The Journal of Physiology, 2010[Dec][15], Vol 588[24], 4871-4889). In the original article, there was an error in the Methods section entitled 'Generation of Epac2 knockout mice' on page 4873. The first sentence of that section should read 'Epac2 KO mice with global disruption of RAPGEF4 gene expression (NCBI GeneID 56508) were generated by the Texas A&M Institute for Genomic Medicine through customized service for Dr. Lu at Louisiana State University Health Sciences Center'. (The following abstract of the original article appeared in record 2011-11969-007). Calcium can be mobilized in pancreatic beta -cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in beta -cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-epsilon (PLC-epsilon ) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-epsilon gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the beta -cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse beta -cells were loaded with the photolabile Ca2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca2+ generated CICR in only 9% of the beta -cells tested, whereas CICR was generated in 82% of the beta -cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A(6-Bnz-cAMP-AM)orEpac2 (8-pCPT-2'-O-Me-cAMP-AM)selectively. However, in beta -cells of PLC-epsilon KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC-epsilon KO beta -cells with recombinant PLC-epsilon rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-epsilon with amutated Ras association (RA) domain, or a H1640L PLC-epsilon that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT beta -cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC-epsilon exists in beta -cells, and that the activities of Epac2 and PLC-epsilon are key determinants of CICR in this cell type.

Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of alpha-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-beta (Abeta)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Abeta yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Abeta42. GFP-Abeta42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Abeta42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Delta), GFP-Abeta42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Abeta42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Abeta42 in wild-type compared to the Atg8Delta mutant. Further, latrepirdine treatment attenuated Abeta42-induced toxicity in wild-type cells but not in the Atg8Delta mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Abeta42.

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2). The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

Disruption of cellular cholesterol balance results in pathologic processes including atherosclerosis, metabolic syndrome, type II diabetes and Alzheimer's disease. Maintenance of cholesterol homeostasis requires constant metabolic adjustment, achieved partly through the fine regulation of the classical transcription factors (e.g., by SREBP and LXR), but also through members of a class of noncoding RNAs termed miRNAs. Some miRNAs have now been identified to be potent post-transcriptional regulators of lipid metabolism genes, including miR-122, miR-33, miR-758, and miR-106b. Different strategies have been developed to modulate miRNA effects for therapeutic purposes. The promise demonstrated by the use of anti-miRs in human preclinical studies, in the case of miR-122, raises the possibility that miR-33, miR-758, and miR-106b may become viable therapeutic targets in future. This review summarizes the evidence for a critical role of some miRNAs in regulating cholesterol metabolism and suggests novel ways to manage dyslipidemias and cardiovascular diseases.

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

We have developed a mouse model of atherosclerotic plaque regression in which an atherosclerotic aortic arch from a hyperlipidemic donor is transplanted into a normolipidemic recipient, resulting in rapid elimination of cholesterol and monocyte-derived macrophage cells (CD68+) from transplanted vessel walls. To gain a comprehensive view of the differences in gene expression patterns in macrophages associated with regressing compared with progressing atherosclerotic plaque, we compared mRNA expression patterns in CD68+ macrophages extracted from plaque in aortic aches transplanted into normolipidemic or into hyperlipidemic recipients. In CD68+ cells from regressing plaque we observed that genes associated with the contractile apparatus responsible for cellular movement (e.g. actin and myosin) were up-regulated whereas genes related to cell adhesion (e.g. cadherins, vinculin) were down-regulated. In addition, CD68+ cells from regressing plaque were characterized by enhanced expression of genes associated with an anti-inflammatory M2 macrophage phenotype, including arginase I, CD163 and the C-lectin receptor. Our analysis suggests that in regressing plaque CD68+ cells preferentially express genes that reduce cellular adhesion, enhance cellular motility, and overall act to suppress inflammation.

The macrophage is exquisitely sensitive to its microenvironment, as demonstrated primarily through in vitro study. Changes in macrophage phenotype and function within the atherosclerotic plaque have profound consequences for plaque biology, including rupture and arterial thrombosis leading to clinical events such as myocardial infarction. We review the evidence for dynamic changes in macrophage numbers and macrophage differentiation within the atherosclerotic plaque microenvironment and discuss potential approaches to target macrophage differentiation for therapeutic benefit in cardiovascular disease.

Biomineralization is a multifactorial and complex process, which results in the deposition of mineral crystals in the extracellular matrix of various tissues. Physiological mineralization is restricted to tissues, such as bones, teeth, and certain areas of cartilage. Pathological or ectopic mineralization can occur in many soft tissues, including articular cartilage, cardiovascular tissues, kidney, ligaments, and tendons, and can lead to serious problems. Therefore, the understanding of factors and mechanisms that regulate the mineralization process is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. This review will discuss some of the mechanisms and factors that regulate physiological mineralization and their potential roles in ectopic mineralization. Finally, potential therapeutic approaches for the treatment of ectopic mineralization are being discussed.

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr'' (KA'') interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA'' cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA'' cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA'' fate specification, but prolonged Hh signaling interferes with KA'' differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA'' cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.