Faculty Bibliography

A role for cystatin C (CysC) in the pathogenesis of Alzheimer's disease (AD) has been suggested by the genetic linkage of a CysC gene (CST3) polymorphism with late-onset AD, the co-localization of CysC with amyloid-beta (Abeta) in AD brains, and binding of CysC to soluble Abeta in vitro and in mouse models of AD. This study investigates the binding between Abeta and CysC in the human central nervous system. While CysC binding to soluble Abeta was observed in AD patients and controls, a SDS-resistant CysC/Abeta complex was detected exclusively in brains of neuropathologically normal controls, but not in AD cases. The association of CysC with Abeta in brain from control individuals and in cerebrospinal fluid reveals an interaction of these two polypeptides in their soluble form. The association between Abeta and CysC prevented Abeta accumulation and fibrillogenesis in experimental systems, arguing that CysC plays a protective role in the pathogenesis of AD in humans and explains why decreases in CysC concentration caused by the CST3 polymorphism or by specific presenilin 2 mutations can lead to the development of the disease. Thus, enhancing CysC expression or modulating CysC binding to Abeta have important disease-modifying effects, suggesting a novel therapeutic intervention for AD

The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding

Purpose: Unlike other DMARDs methotrexate diminishes the risk of Atherosclerotic Cardiovascular Disease (ASCVD) in patients with Rheumatoid Arthritis and adenosine, acting at adenosine A2A receptors, has been shown to mediate the anti-inflammatory effects of methotrexate. Adenosine inhibits the first step in formation of atherosclerotic plaque, foam cell formation in macrophages and this effect appears to be mediated by enhanced expression of cholesterol 27-hydroxylase, an enzyme involved in reverse cholesterol transport. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by apoE or 27-hydroxylase (27OH'ase), proteins involved in reverse cholesterol transport. Method: THP-1 cells, a human monocytoid cell line, were infected with lentiviral vectors expressing siRNA for either apoE or 27OH'ase or scrambled RNA and infected cell lines were selected by incubation with puromycin. Foam cell formation was induced in THP-1 cells by incubation with interferon- (500U/ml) and % foam cells enumerated in 5 high power fields. 3H-Cholesterol efflux was measured after loading with label. Results: Specific lentiviral siRNA infection markedly reduces apoE (p< 0.0001, apoE siRNA vs. control, n=3) or 27OH'ase mRNA (p< 0.0001, 27-hydroxylase siRNA vs. control, n=3) and protein (p< 0.0107, 27-hydroxylase siRNA vs. control n= 3) in THP-1 cells. Despite diminished apoE expression CGS-21680 (1muM), an adenosine A2A receptor agonist, inhibits IFN-induced foam cell formation (p< 0.0002, IFN CGS vs. IFN alone, n= 4) but has no effect on foam cell formation in 27OH'ase KD cells. CGS21680 increases cholesterol efflux in wild type and apoE1 KD cells (from 9.5% to 17.5+2.5% and from 10.0+2% to 17.5 +2%, respectively) but not 27OH'ase KD cells. Conclusion: Adenosine A2A receptor-mediated increases in reverse cholesterol transport leading to diminished foam cell formation explains the anti-atherosclerotic effects of methotrexate

In recent years, stable isotope labeling by amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomic method. In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched. SILAC is a powerful tool for the study of intracellular signal transduction. In particular, it has been very popular and successful in quantitative analysis of phosphoty-rosine (pTyr) proteomes to characterize pTyr-dependent signaling pathways. In this chapter, we describe the SILAC procedure and use EphB signaling pathway as an example to illustrate the use of SILAC to investigate such pathways

Invasive aspergillosis remains difficult to diagnose despite advances in imaging and antigen-based serological testing. To overcome this problem, nucleic acid (NA)-based amplification assays were introduced to identify infecting pathogens. Unfortunately, the reliability of such assays to detect Aspergillus spp. has met with mixed success. A new generation of NA platforms are emerging, which greatly improve our ability to detect Aspergillus-specific DNA and RNA from respiratory and blood samples. These platforms can accurately detect a single genome, and the emergence of pan-fungal and pan-Aspergillus probes offer promise for broader detection. PCR remains the most important platform, especially when coupled with real-time probes. It is multiplex friendly and can distinguish between closely related target sequences. Nucleic acid sequence-based amplification (NASBA) is an RNA-directed isothermal transcription-based amplification platform, which is more robust than PCR resulting in a 10(14)-fold amplification. RNA-based detection facilitates more target options and can be used to assess cell viability. Both DNA and RNA amplification platforms take advantage of allele-specific properties of probes, which are valuable for assessing drug resistance markers. Finally, as new molecular diagnostic platforms mature, their role may expand to include early monitoring of therapy.

A strong link is indicated between cardiovascular disease (CVD) and risk for developing Alzheimer's disease (AD), which may be exacerbated by the major AD genetic risk factor apolipoprotein Eepsilon4 (APOEepsilon4). Since subjective memory complaint (SMC) may potentially be an early indicator for cognitive decline, we examined CVD risk factors in a cohort of SMC. As amyloid-beta (Abeta) is considered to play a central role in AD, we hypothesized that the CVD risk profile (increased LDL, reduced HDL, and increased body fat) would be associated with plasma Abeta levels. We explored this in 198 individuals with and without SMC (average age = 63 years). Correlations between Abeta40 and HDL were observed, which were stronger in non-APOEepsilon4 carriers (rho = -0.315, p < 0.001) and in SMC (rho = -0.322, p = 0.01). There was no relationship between percentage body fat and Abeta40 in this cohort. Age and HDL remained predictive for plasma Abeta40 using multivariate regression analysis. We report a novel negative association between HDL and Abeta, which if demonstrated to be causal has implications for the development of lifestyle interventions and/or novel therapeutics. The relationship between HDL and Abeta and the potential significance of such an association needs to be validated in a larger longitudinal study.

BACKGROUND: The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied. METHODOLOGY/PRINCIPAL FINDINGS: We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (microCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13. CONCLUSION: A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.