Faculty Bibliography

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

Introduction: Atherosclerosis is an inflammatory disease. Its major clinical manifestation, coronary artery disease, is the leading cause of death in the western world. The disease is caused by the rupture of macrophage-laden and highly-inflamed atherosclerotic plaques, which are prone to rupture and cause myocardial infarctions. Oral statin therapy is widely used to reduce blood cholesterol levels in patients with atherosclerosis, and it is believed to have modest anti-inflammatory effects. We previously developed a strategy that aims at amplifying these anti-inflammatory effects through statin delivery to plaque macrophages using a reconstituted high density lipoprotein (rHDL) nanoparticle as a delivery vehicle. This statin rHDL ([S]-rHDL) nanotherapy reduced plaque macrophages by 80% (Supplementary). In the current study, we set out a novel nanomedical treatment paradigm, based on the aforementioned [S]-rHDL nanotherapy, which aims at realizing rapid remodeling of advanced atherosclerotic plaques towards a favorable phenotype in Apolipoprotein E-/- (ApoE KO) mice. Design and Results: ApoE KO mice received 26 weeks of high-cholesterol diet to develop advanced atherosclerotic plaques. After the diet, the mice first received high dose [S]-rHDL for a week (60 mg/kg simvastatin, 4 intravenous injections /week), followed by either low dose [S]-rHDL (15 mg/kg simvastatin, 2 intravenous injections /week), oral simvastatin (15 mg/kg/day), or no treatment for another 8 weeks (A). We evaluated the efficacies of the therapies during the course of the 9-week treatment with a MATLAB-assisted histological assessment of aortic roots, and CD68 immunostaining and hematoxylin phloxine saffron stain (HPS)) were performed (B). In vivo magnetic resonance imaging (MRI) of abdominal aortas and blood tests were done as well. After one week high dose [S]-rHDL treatment, macrophage levels in aortic roots were reduced by 70% (P < 0.001). The low levels were maintained by a subsequent 8-week low dose [S]-rHDL (48 % lowe!

High Density Lipoprotein (HDL) is a natural nanoparticle involved in the transport of cholesterol throughout the body. HDL has been shown to exhibit atheroprotective properties as it promotes cholesterol efflux from atherosclerotic plaque macrophages in the arterial wall. Various laboratories have focused on the reconstitution of HDL (rHDL) for a variety of reasons, ranging from a better understanding of the structural biology of apolipoproteins to the use of rHDL as an injectable therapeutic (1). A recent effort centers around the use of rHDL as a natural nanoparticle platform for the delivery of contrast agents such as gadolinium chelates, iron oxide or gold nanoparticles, and employing them as molecular imaging contrast agents (2). To date, multistep production protocols pose a limit on the synthesis of batch quantities and are sensitive to inter-batch variations. In order to scale up the production process and to judiciously control rHDL's composition we have developed a modular single-step approach based on recently introduced microfluidics technology (3) that enables the standardized mass production of such lipoprotein-based nanoparticles. Materials and methods Organic solutions containing phospholipids and imaging agents (QD, FeO-NP, Au-NP, DiO) were injected into a microfluidic chip alongside an aqueous solution containing ApoA1. Within the chip the controlled flow streams generate microvortices where fast mixing of the solutions leads to the instantaneous formation of HDL-like nanoparticles (Figure 1). HDL particles produced by this microfluidics method, which we refer to as muHDL, had similar physicochemical properties (size, morphology) to particles produced by conventional methods and natural HDL. Moreover cell based assays demonstrated that muHDL nanoparticles displayed a similar bioactivity profile to natural HDL. muHDL that encapsulated hydrophobic dies (DiO) or nanocrystals such as quantum dots (QD), gold (Au) or iron oxide (FeO) nanoparticles were characterized and evaluated in!

Introduction 2nd generation polymeric nanoparticles have shown significant advantages in drug delivery. They can be loaded with poorly water soluble drugs,1 their size can be judiciously controlled2 and their surface can be functionalized with a PEG coating and/or targeting ligands. Importantly, the polymeric core can be loaded with drugs and/or contrast agents for which the release rates can be controlled by the choice of polymer composition and molecular weight. High-density lipoprotein (HDL) is a natural nanoparticle that transports fats through the body, which has an inherent affinity for atherosclerotic plaques. HDL-like nanoparticles labeled with contrast agents have been shown suitable for molecular imaging as they effectively target atherosclerotic plaque. In the current study we developed a novel HDL-like hybrid nanoparticle using recently developed microfluidics technology.2 The nanoparticle is comprised of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core suitable for the delivery of drugs in a controlled manner. The versatility of the approach also allows the incorporation of functional lipids to render multifunctional nanoparticles with imaging, therapeutic and atherosclerosis targeting properties. Methods and Results Hybrid polymer-HDL nanoparticles with a PLGA core and a coating comprised of lipids and apolipoprotein A1 (PLGA-HDL) were synthesized using microfluidics. The synthetic approach consists of the rapid injection of the components in three different channels of a microfluidics chip. Amphiphilic phospholipids and PLGA were dissolved in a mixture of ethanol and acetonitrile. This solution was injected in the middle channel of the microfluidic chip and mixed with an aqueous apoprotein A1 (ApoA1) solution injected in the two outer channels. Inside the chip, controlled nanoprecipitation occurred through microvortices, resulting in the instantaneous and continuous production of hybrid PLGA-HDL nanoparticles with high reproducibility (!

Tumor necrosis factor-a (TNFa) has received the greatest attention because of its position at the apex of the pro-inflammatory cytokine cascade, and its dominance in the pathogenesis of inflammation. Anti-TNF therapies have been accepted as the effective approach to treating rheumatoid arthritis. In a global screen for the binding proteins of progranulin (PGRN), an autocrine growth factor with multiple functions, we found that PGRN bound to TNFR1 and TNFR2. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. More importantly, Atsttrin, an engineered protein composed of three PGRN fragments, exhibited potent anti-inflammatory activity, which surpassed PGRN itself, in vivo. This talk will focus on the interplay between PGRN and TNF in inflammatory arthritis as well as the immunological mechanism involved. The talk will also highlight Atsttrin. Identification of PGRN as a ligand of TNFR and an antagonist of TNFalpha signaling not only betters our understanding of the pathogenesis of inflammatory arthritis, but also provides new therapeutic interventions for various TNFalpha-mediated pathologies and conditions, including rheumatoid arthritis

Alzheimer disease (AD) is a form of neurodegeneration that develops over the course of multiple decades and as a result of the accumulation of the pathogenic amyloid-beta (Abeta) peptide, also known as A4. In late-stage AD, failure of autophagic clearance results in neuronal cell bodies that are almost entirely consumed by autophagic vacuoles (AVs). Previously, we have shown that the potential AD drug latrepirdine (aka Dimebon((R))), a Russian antihistamine that has shown mixed results in phase II clinical trials in AD, regulates metabolism of the amyloid-beta/A4 precursor protein (APP). In two Molecular Psychiatry papers in 2012, we sought to determine the mechanism through which latrepirdine regulates APP metabolism and to determine, using an Alzheimer mouse model, whether latrepirdine provides protection from the toxicity associated with the accumulation of Abeta. In cultured cells, we provided evidence that latrepirdine stimulates MTOR- and ATG5-dependent autophagy, leading to the reduction of intracellular levels of APP metabolites, including Abeta. Consistent with this finding, we found that chronic latrepirdine administration resulted in increased levels of the biomarkers thought to correlate with autophagy activation in the brains of TgCRND8 (APP K670M, N671L, V717F) or wild-type mice, and that treatment was associated with abrogation of behavioral deficit, reduction in Abeta neuropathology, and prevention of autophagic failure among TgCRND8 mice.