ABSTRACT: Predisposition to Alzheimer's disease (AD) may arise from lipid metabolism perturbation, however, the underlying mechanism remains elusive. Here, we identify ATAD3A, a mitochondrial AAA-ATPase, as a molecular switch that links cholesterol metabolism impairment to AD phenotypes. In neuronal models of AD, the 5XFAD mouse model and post-mortem AD brains, ATAD3A is oligomerized and accumulated at the mitochondria-associated ER membranes (MAMs), where it induces cholesterol accumulation. Suppressing ATAD3A oligomerization by heterozygous ATAD3A knockout normalizes brain cholesterol turnover and MAM integrity, suppresses APP processing and synaptic loss, and consequently reduces AD neuropathology and cognitive deficits in AD transgenic mice. These findings reveal a role for ATAD3A oligomerization in AD pathogenesis and suggest ATAD3A as a potential therapeutic target for AD.
Project description:An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (3 normal and 3 increased). Three of the six cell lines were capable of generating tumors and tumor formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially-expressed between the tumorigenic and non-tumorigenic cell lines. Frizzled-6 was up-regulated to the greatest extent (7.78 fold) in tumorigenic cell lines compared to non-tumorigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumor-initiating cells and poor prognosis for other tumors. The increased expression of frizzled-6 was confirmed by QPCR and Western blot analysis. Additionally, the tumorigenic cell lines also had an increase in the percentage of side population cells compared to non-tumorigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumorigenicity, frizzled-6, or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumorigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumorigenesis and tumor-initiating cells. A total of six canine primary osteosarcoma cell lines were used in this study. Three cell lines were capable of forming tumors when transplanted into mice (tumorigenic) and three cell lines were not capable of forming tumors upon transplant into mice (non-tumorigenic). The gene expression data is from the primary cell lines, not the transplanted cells. There were no reference cell lines or controls used in this study.
Project description:This work presents improved protease digestion conditions for membrane protein detection. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen and applied to proteins from Escherichia coli. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.
Project description:Discrepancies in blood sample collection and processing could have a significant impact on levels of peptides in the blood, thus sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several pre-analytical processing conditions, including different storage times and temperatures of blood or plasma samples and different centrifugation forces, on the levels of peptides in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Both time and temperature were identified as major factors for peptide variation.
Project description:Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.
Project description:The early oligomerization of amyloid ?-protein (A?) has been shown to be an important event in the pathology of Alzheimer's disease (AD). Designing small molecule inhibitors targeting A? oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on A? self-assembly. CLR01 was found to bind to A? directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of A?42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early A? oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and A? oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment.
Project description:Levels of membrane-associated cholesterol were shown to be increased in the brain of individuals with sporadic Alzheimer’s disease (AD) and correlated with the severity of the disease. We previously found that heavy membrane cholesterol burden promotes amyloid precursor protein (APP) endocytosis and processing, leading to increased amyloid-A) secretion. We hypothesized that such an increase of cholesterol could trigger sporadic AD. We thus acutely loaded the plasma membrane of neurons in culture with cholesterol to reach the 30 % increase observed in AD brains. We showed by multiplex electro-chemiluminescence immuno-assay that transient membrane cholesterol loading produced a significant increase of A42 secretion. We also found that early endosomes were enlarged and more prone to aggregation using confocal and electron microscopy and that APP vesicular transport in neuronal processes was slowed down using fluorescence live-imaging. In addition, treatment of neurons with cholesterol induced changes in gene expression profile that are reminiscent of early AD. This model of membrane cholesterol increase in cultured neurons reproduces most of early AD changes and could thus be relevant for deciphering early mechanisms and design new targets for sporadic AD. In this study, we loaded the plasma membrane of neurons with 30% more cholesterol and observed the effects on gene expression. We compared gene expression of primary hippocampal neurons treated or not with cholesterol using 4 independant replicates in each group.
Project description:The formation of small A?42 oligomers has been implicated as a toxic species in Alzheimer disease (AD). In strong support of this hypothesis we found that overexpression of Yap1802, the yeast ortholog of the human AD risk factor, phosphatidylinositol binding clathrin assembly protein (PICALM), reduced oligomerization of A?42 fused to a reporter in yeast. Thus we used the A?42-reporter system to identify drugs that could be developed into therapies that prevent or arrest AD. From a screen of 1,200 FDA approved drugs and drug-like small compounds we identified 7 drugs that reduce A?42 oligomerization in yeast: 3 antipsychotics (bromperidol, haloperidol and azaperone), 2 anesthetics (pramoxine HCl and dyclonine HCl), tamoxifen citrate, and minocycline HCl. Also, all 7 drugs caused A?42 to be less toxic to PC12 cells and to relieve toxicity of another yeast AD model in which A?42 aggregates targeted to the secretory pathway are toxic. Our results identify drugs that inhibit A?42 oligomers from forming in yeast. It remains to be determined if these drugs inhibit A?42 oligomerization in mammals and could be developed as a therapeutic treatment for AD.
Project description:Amyloid-? (A?) oligomers largely initiate the cascade underlying the pathology of Alzheimer's disease (AD). Galectin-3 (Gal-3), which is a member of the galectin protein family, promotes inflammatory responses and enhances the homotypic aggregation of cancer cells. Here, we examined the role and action mechanism of Gal-3 in A? oligomerization and A? toxicities. Wild-type (WT) and Gal-3-knockout (KO) mice, APP/PS1;WT mice, APP/PS1;Gal-3<sup>+/-</sup> mice and brain tissues from normal subjects and AD patients were used. We found that A? oligomerization is reduced in Gal-3 KO mice injected with A?, whereas overexpression of Gal-3 enhances A? oligomerization in the hippocampi of A?-injected mice. Gal-3 expression shows an age-dependent increase that parallels endogenous A? oligomerization in APP/PS1 mice. Moreover, A? oligomerization, Iba1 expression, GFAP expression and amyloid plaque accumulation are reduced in APP/PS1;Gal-3<sup>+/-</sup> mice compared with APP/PS1;WT mice. APP/PS1;Gal-3<sup>+/-</sup> mice also show better acquisition and retention performance compared to APP/PS1;WT mice. In studying the mechanism underlying Gal-3-promoted A? oligomerization, we found that Gal-3 primarily co-localizes with Iba1, and that microglia-secreted Gal-3 directly interacts with A?. Gal-3 also interacts with triggering receptor expressed on myeloid cells-2, which then mediates the ability of Gal-3 to activate microglia for further Gal-3 expression. Immunohistochemical analyses show that the distribution of Gal-3 overlaps with that of endogenous A? in APP/PS1 mice and partially overlaps with that of amyloid plaque. Moreover, the expression of the A?-degrading enzyme, neprilysin, is increased in Gal-3 KO mice and this is associated with enhanced integrin-mediated signaling. Consistently, Gal-3 expression is also increased in the frontal lobe of AD patients, in parallel with A? oligomerization. Because Gal-3 expression is dramatically increased as early as 3 months of age in APP/PS1 mice and anti-A? oligomerization is believed to protect against A? toxicity, Gal-3 could be considered a novel therapeutic target in efforts to combat AD.
Project description:As an important neuropathological hallmark of Alzheimer's disease (AD), the oligomerization of amyloid-? (A?) peptides has been intensively investigated in both theoretical and experimental studies. However, the oligomerization space in terms of the kinetics, molecular mechanism, and oligomer structures remains mysterious to us. An equation that can quantitatively describe the time it takes for A? oligomers to appear in the human brain at a given A? monomer concentration is extremely vital for us to understand the development and disease progression of AD. In this study, we utilized molecular dynamics (MD) simulations to investigate the oligomerization of A?42 peptides at five different monomer concentrations. We have elucidated the formation pathways of A? tetramers, characterized the oligomer structures, estimated the oligomerization time for A? dimers, trimers, and tetramers, and for the first-time derived equations that could quantitatively describe the relationship between the oligomerization time and the monomer concentration. Applying these equations, our prediction of oligomerization time agrees well with the experimental and clinical findings, in spite of the limitations of our oligomerization simulations. We have found that the A? oligomerization time depends on the monomer concentration by a power of -2.4. The newly established equations will enable us to quantitatively estimate the risk score of AD, which is a function of age. Moreover, we have identified the most dominant pathway of forming A? tetramers, probably the most important and toxic A? oligomer. Our results have shown that the structures of A?42 dimer, trimer, and tetramer, which are distinguishable from each other, depend on the monomer concentration at which the oligomers form. Representative oligomer structures, which can serve as potential drug targets, have been identified by clustering analysis. The MD sampling adequacy has been validated by the excellent agreement between the calculated and measured collisional cross section (CCS) parameters (the prediction errors are within 2%). In a conclusion, this study provides the kinetics and structure basis for developing inhibitors to decelerate the A? oligomerization process.
Project description:In this study, we demonstrate the use of Molecular topology (MT) in an Alzheimer's disease (AD) drug discovery program. MT uses and expands upon the principles governing the molecular connectivity theory of numerically characterizing molecular structures, in the present case, active anti-AD drugs/agents, using topological descriptors to build models. Topological characterization has been shown to embody sufficient molecular information to provide strong correlation to therapeutic efficacy.We used MT to include multiple bioactive properties that allows for the identification of multi-functional single agent compounds, in this case, the dual functions of ?-amyloid (A?) -lowering and anti-oligomerization. Using this technology, we identified and designed novel compounds in chemical classes unrelated to current anti-AD agents that exert dual A? lowering and anti-A? oligomerization activities in animal models of AD. AD is a multifaceted disease with different pathological features.Our study, for the first time, demonstrated that MT can provide novel strategy for discovering drugs with A? lowering and anti-aggregation dual activities for AD.