Faculty Bibliography

OBJECTIVES: Alzheimer's disease (AD) is the leading cause of dementia and the most common form of amyloidosis in humans. Extensive extracellular deposition of amyloid-beta (Abeta), a 40-42 amino acid degradation product of APP, is considered a hallmark feature of AD. Our attention is focused on the highly heterogeneous biochemical nature of the brain Abeta species. METHODS AND POPULATION: We have fractionated water-soluble, detergent-soluble and formic acid-solube Abeta species from transgenic mouse models of amyloid deposition and AD cases. Subsequently, we applied a combination of biochemical techniques including immunoprecipitation followed by identification of Abeta fragments with a novel highly sensitive matrixassisted laser desorption/ionization time-of-flight mass spectrometry technique. RESULTS: Our biochemical data on the Abeta species present in sporadic and familial AD cases and in transgenic mouse models highlight the presence of similar N- and C-terminally truncated fragments-likely reflecting the ability of multiple proteases to degrade Abeta in situ- and several post-translational modifications with still unclear roles in the amyloidogenesis mechanism. Notably, not all the brain Abeta peptides have identical solubility properties. SIGNIFICANCE OF STUDY: In view of these findings and the growing evidence that an imbalance between Abeta production and clearance plays a major role in the process of Abeta deposition, we hypothesize that certain truncated and post-translationally modified Abeta fragments have a distinct and opposite role in clearance and fibrillization mechanisms and that the spectrum and abundance of these species vary according to the magnitude of the amyloid load

The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important implications for understanding the mechanical dysfunction and cardiac arrhythmias that accompany alterations of ID architecture

ABSTRACT: INTRODUCTION: Multi-marker molecular assays have impacted management of early stage breast cancer, facilitating adjuvant chemotherapy decisions. We generated prognostic models that incorporate protein-based molecular markers and clinico-pathological variables to improve survival prediction. METHODS: We used a quantitative immunofluorescence method to study protein expression of 14 markers included in the Oncotype DX assay on a 638 breast cancer patient cohort with 15-year follow-up. We performed cross-validation analyses to assess performance of multivariate Cox models consisting of these markers and standard clinico-pathological covariates, using an average time-dependent Area Under the Receiver Operating Characteristic curves and compared it to nested Cox models obtained by robust backward selection procedures. RESULTS: A prognostic index derived from of a multivariate Cox regression model incorporating molecular and clinico-pathological covariates (nodal status, tumor size, nuclear grade, and age) is superior to models based on molecular studies alone or clinico-pathological covariates alone. Performance of this composite model can be further improved using feature selection techniques to prune variables. When stratifying patients by Nottingham Prognostic Index (NPI), the most prognostic markers in high and low NPI groups differed. Similarly, for the node-negative, hormone receptor-positive sub-population, we derived a compact model with three clinico-pathological variables and two protein markers that was superior to the full model. CONCLUSIONS: Prognostic models that include both molecular and clinico-pathological covariates can be more accurate than models based on either set of features alone. Furthermore, feature selection can decrease the number of molecular variables needed to predict outcome, potentially resulting in less expensive assays

Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microscopy

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) to enable cells to recover from ER stress. If the UPR fails to restore normal ER function, apoptosis is induced instead. We have demonstrated that melanocytes have the capacity to adapt to chronic ER stress and escape from UPR-induced cell death. Mutations in the pinkeyed dilution/oculocutaneous albinism type 2 (Oca2)-gene result in altered tyrosinase processing and trafficking in melanocytes, accompanied by accumulation of misfolded tyrosinase in the ER. Despite this chronic overload of ER-retained tyrosinase, Oca2-mutant melanocytes do not show diminished viability suggesting that they have adapted to the ER stress. The aim of this study is to elucidate the process of adaptation to ER stress in melanocytes. Differential protein expression between wildtype (melan-a) and Oca2-melanocytes (melan-p) was analyzed using microarray assays and Western blotting. Adaptation to chronic ER stress was studied by comparing wildtype murine melanocytes dosed with the ER stressor thapsigargin to Oca2-mutants. We observed increased Ire1 expression in Oca2-melanocytes compared to wildtype, indicating that this UPR pathway is activated. Prolonged Ire1 signaling after UPR activation in stressed cells has been found to promote cell survival. In addition, expression of proteins involved in the pro-apoptotic Perk pathway appears to be decreased in Oca2- melanocytes. However, Oca2-mutants also demonstrated up-regulation of Chop, which typically promotes cell death by down-regulating expression of anti-apoptotic Bcl-2. Remarkably, the pro-apoptotic effects of Chop expression appear to be mitigated by up-regulation of Bcl-2 expression. Furthermore, expression of pro-apoptotic proteins such as Bid and caspase 1 were down-regulated in Oca2-mutant melanocytes as compared to wildtype cells. Acute ER stress (0-24 h) in wildtype melanocytes activated all three UPR pathways, but Ire1 signaling was attenuated in chronically stressed cells (12 days), while Perk signaling was maintained. Cell viability did not change during this period. Thus, wildtype melanocytes adapted to chronic ER stress despite sustained activation of pro-apoptotic pathways. These results indicate that chronically stressed wildtype melanocytes and Oca2-mutant melanocytes adapt to ER stress by differential mechanisms. Melanocytes may thus adapt to ER-stress by multiple mechanisms, some of which may account for the increased drug resistance observed in melanocytes and melanoma

Background: Accumulation of immature proteins in the Endoplasmic Reticulum (ER) causes organelle stress that is counteracted by the unfolded protein stress response (UPR). Three pathways compose the UPR and are initiated when Ire1, Perk and Atf6 respectively, are released from heterodimers formed with the ER chaperone BiP. The UPR signals down-regulation of global translation and increased ER-chaperone expression. Ire1 is phosphorylated, activating its nuclease activity, which leads to splicing of the X-box binding protein 1 (Xbp1). The spliced RNA encodes a transcription factor that regulates expression of a subset of genes that comprise one arm of the UPR. Apoptosis is initiated if homeostasis is not re-established following UPR activation. The Ire1 pathway has recently been shown to play arole in the development of vitiligo, while the UPR has been implicated in keratinocyte response to UVB and in drug resistance in melanoma. We therefore investigated the melanocyte response to ER stress induced by chemical ER disruptors and by oxidative stress (the mechanism by which we propose UV exposure perturbs the melanocyte ER). Method: Wild-type mouse melanocytes were treated with thapsigargin, which disrupts the calcium balance in the ER causing UPR induction, and cells harvested at 6, 12 and 24 h. Western blot and microarray analyses were performed and data evaluated to identify pathways activated by thapsigargin treatment. In addition, melanocytes were dosed with compounds that induce oxidative stress. RNA was harvested and evaluated for activation of the UPR by Xbp-1 splicing. Results: IRE1 expression was upregulated within 6 h of treatment with thapsigargin, which promoted splicing of XBP1 mRNA and activation of its transcription factor activity. PERK and its downstream target CHOP were phosphorylated and HA-tagged ATF6 was cleaved within 6-12 h of treatment. Up-regulation of BiP and ER chaperones such as Ero1 and down-regulation of tyrosinase were also observed. In addition, several p53-related pathways were modulated in response to thapsigargin. Induction of oxidative stress was found to induce Xbp1 splicing. Conclusions: The UPR may play an important role in melanocyte response to stress, including response to oxidative stress induced by UV exposure. Dysfunction of this response may contribute to initiation and progression of vitiligo and drug resistance in melanoma

Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.