Faculty Bibliography

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

Patients from the Indian subcontinent have a distinct cardiovascular risk profile with profound health consequences. South Asians tend to develop more severe coronary artery disease at a younger age, and may also suffer from earlier myocardial infarction and heart failure. The genesis of this risk is multi-factorial. One important culprit is increased insulin resistance, possibly due to recently identified genetic polymorphisms. Another possible explanation is subclinical inflammation and a prothrombotic environment, as evidenced by increased levels of homocysteine, plasminogen activator inhibitor-1, and fibrinogen. The lipid profile of South Asians may play a role, as this population is known to have elevated levels of lipoprotein (a), as well as lower levels of HDL. In addition, this HDL may be dysfunctional, as this population may have a higher prevalence of low levels of HDL2b, as well as an increased preponderance of smaller HDL. Current guidelines for primary and secondary prevention have not reflected our growing insight into the unique characteristics of the South Asian population, and may need to evolve to reflect our knowledge

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins.

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

Purpose: Survival of bone implants depends on biological fixation, and prosthesis loosening can be catastrophic leading to replacement of prostheses. Inflammation and osteoclast-mediated bone resorption in response to wear particles near prostheses contribute to loosening. Because we have demonstrated that adenosine A_2Areceptor activation is anti-inflammatory and prevents osteoclast formation and function we hypothesized that adenosine A_2Areceptor agonists might prevent osteoclast-mediated bone resorption at the site of prosthesis wear in a calvarial model of wear particle-induced bone resorption. Methods: Eighteen C57Bl/6 mice age 6-8 weeks were anesthetized by intraperitoneal injection of ketamine and xylazine and a 1cm midline sagittal incision was made over the calvarium anterior to the line connecting both ears. Six animals received no particles (control), and 12 received 15 ml of polyethylene particle suspension. Of the 12 mice receiving particulate, 6 were injected subcutaneously at the surgical site with 20 ml of 10 muM CGS21680 (A_2Areceptor agonist), and 6 mice were injected with saline 0.9%, beginning immediately after incision closure and continuing every other day until sacrifice. Animals were sacrificed after 14 days and the calvaria were removed, fixed, and prepared for microCT and histological staining with TRAP. Results: Histologic examination of calvaria demonstrated lymphocytic infiltration in both particle-exposed groups. TRAP staining revealed a reduction in osteoclast differentiation after treatment with CGS21680. mCT showed pitting and increased porosity in both particle-exposed groups compared to controls, although in CGS21680-treated mice the reduction in cortical bone was significantly less than in the untreated particle-exposed mice (p<0.01). Control bone volume/trabecular volume was significantly greater (p<0.005) than in either particulate group, however, calvarial bone from CGS21680-treated mice had significantly greater mean bone volume than did the untreated group (p<0.0005). Trabecular thickness was significantly reduced in both CGS21680-treated and untreated particle-exposed groups as compared to control mice (p<0.05). Finally, digital morphometric analysis of microCTs reveals that CGS21680 significantly reduced the area of bone pitting compared to control particle-treated mice (p<0.05). (Table presented) Conclusions: Adenosine A_2Areceptor activation reduces inflammation and bone destruction due to prosthetic wear particles. This observation suggests that delivery of an adenosine A_2Aagonist in the cement may enhance orthopedic implant survival, delaying or eliminating the need for revision arthroplasty surgery

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.

The epsilon4 allele of apolipoprotein E (APOE) is currently the major genetic risk factor identified for Alzheimer's disease (AD). Previous in vivo data from our laboratory has demonstrated that amyloid-beta (Abeta) is rapidly removed from the plasma by the liver and kidney and that the rate of its clearance is affected by ApoE in C57BL/6J and APOE-/- mice. To expand upon these findings, we assessed the peripheral clearance of human synthetic Abeta42 in APOE epsilon2, epsilon3, and epsilon4 knock-in and APOE knock-out mice injected with lipidated recombinant apoE2, E3, and E4 protein. Our results show that APOE does influence the rate at which the mice are able to clear Abeta42 from their bloodstream. Both APOE epsilon4 mice and APOE knock-out mice treated with lipidated recombinant apoE4 demonstrated increased retention of plasma Abeta42 over time compared to APOE epsilon2/APOE knock-out rE2 and APOE epsilon3/APOE knock-out rE3 mice. These findings suggest that the peripheral clearance of Abeta42 is significantly altered by APOE genotype. Given that APOE epsilon4 is a risk factor for AD, then these novel findings provide some insight into the role of ApoE isoforms on the peripheral clearance of Abeta which may impact on clearance from the brain

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

Understanding how cells are assembled in three dimensions to generate an organ, or a whole organism, is a pivotal question in developmental biology. Similarly, it is critical to understand how adult stem cells integrate into an existing organ during regeneration or in response to injury. Key to discovering the answers to these questions is being able to study the various behaviors of distinct cell types during development or regeneration. Fate mapping techniques are fundamental to studying cell behaviors such as proliferation, movement, and lineage segregation, as the techniques allow precursor cells to be marked and their descendants followed and characterized over time. The generation of transgenic mice, combined with the use of site-specific recombinases (SSR) in the mouse genome, has provided a means to develop powerful genetic fate mapping approaches. A key advantage of genetic fate mapping is that it allows cells to be genetically marked, and therefore the mark is transmitted to all the descendants of the initially marked cells. By making modifications to the SSRs that render their enzymatic activity inducible, and the development of an assortment of reporter alleles for marking cells, increasingly sophisticated genetic fate mapping studies can be performed. In this chapter, we review the four main genetic fate mapping methods that utilize intrachromosomal recombination to mark cells (cumulative, inducible, clonal, and intersectional) and one interchromosomal method, the tools required to carry out each approach, and the practical considerations that have to be taken into account before embarking on each type of genetic fate mapping study

Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR experiments revealed an upregulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of people with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Results indicate selective upregulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD