Dissertations / Theses on the topic 'Amyloid beta-protein precursor'
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1
Dooley, Nora P. "Analysis of beta-amyloid precursor protein in Alzheimer's fibroblasts." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56982.
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One of the hallmarks of Alzheimer's disease is the accumulation of beta-amyloid protein in the core of neuritic plaques. This 38-40 amino acid polypeptide is derived from a larger precursor known as beta-amyloid precursor protein (BAPP). In this thesis the expression of this precursor in cultured human fibroblasts obtained from Alzheimer's patients and age-matched controls is examined at the mRNA and protein level. Using the technique of reverse transcriptase-polymerase chain reaction, human fibroblasts were found to express BAPP transcripts encoding 770, 751, 714, and 695 amino acids. In immunocytochemical studies employing a monoclonal antibody to BAPP, this precursor was determined to be associated with the intermediate filament network. As well, on Western blots, aside from the bands of predicted molecular weights, a Triton-soluble 57 kDn band was detected. These findings lend support to the theory that aside from its putative extracellular functions, BAPP may play a vital intracellular role.
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Stephens, David John. "Intracellular processing of the Alzheimer's #beta#-amyloid precursor protein." Thesis, St George's, University of London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362427.
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Jung, Sonia Sun-Yung. "Expression and processing of the Alzheimer's beta-amyloid precursor protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0031/NQ64584.pdf.
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Jung, Sonia Sun-Yung 1968. "Expression and processing of the Alzheimer's beta-amyloid precursor protein." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36618.
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Alzheimer's disease (AD) is the most common neurodegenerative disease affecting the elderly. One of the main pathological hallmarks of AD is abundant senile plaques found in brain parenchyma and in the meningovasculature. The core of these senile plaques is predominantly composed of the 40--43 amino acid beta-amyloid (Abeta) peptide which arises from proteolytic cleavage of the beta-amyloid precursor protein (betaAPP), a glycoprotein with a predicted structure of a transmembrane protein. Most studies of betaAPP expression and processing have focused on cultured cell systems. Previously, our laboratory reported that immediately ex vivo human peripheral blood lymphocytes did not express cell surface betaAPP. Here, we investigated expression of betaAPP in various immediately ex vivo cell types from rodent and human species to determine whether or not our previous findings extended to different cell types and species. Cell surface expression of betaAPP was detected on immediately ex vivo rodent and human adult brain cells, and human peripheral blood monocytes by using various anti-betaAPP antibodies and flow cytometry; however, peripheral cells such as rodent lymphoid cells, hepatocytes, and kidney cells or human lymphoid cells did not display cell surface immunoreactivity, although all cells expressed abundant betaAPP intracellularly. This suggests cell-specific processing of the betaAPP molecule. We extended our studies with human peripheral blood monocytes to examine any differences between different age groups and AD patients. We demonstrate that cell surface betaAPP decreases with aging; however, it is significantly increased in AD patients compared to healthy age-matched controls. Our data suggest that a proportion of peripheral Abeta may be derived from monocyte/macrophages, and that defects in brain cell processing of betaAPP in AD may be shared by this readily accessible peripheral cell. Investigation of immediately ex vivo human adult brain cells demonstrat
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Kim, Joung-Hun. "Electrophysiological and biochemical studies of #beta#-amyloid precursor protein fragments." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394383.
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Sultana, Joynab. "Behavioral Effects of Amyloid Precursor Protein beta Mutation in zebrafish." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-421155.
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Amyloid precursor protein beta (βAPP) plays an important role in the pathogenesis of Alzheimer’s disease. An appb mutant strain of zebrafish has been previously generated and has shown increased boldness. Here we tested boldness by Novel Tank Diving Test and compared the results between the wildtype AB strain controls (WT) (N=16) and appb mutant strain (N=28), as well as between two Swedish testing institutions that use different protocols. Fish were tracked by automated video tracking in Ethovision. Compared with the wild type fish, using both the Uppsala and Gothenburg protocols, the mutant fish have a higher cumulative duration in the top area suggesting increased boldness. Greater boldness in mutants appears specifically context dependent and only expressed when the test fish is taken from a larger group of fish.
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Selivanova, Alexandra. "Intracellular dynamics of Alzheimer disease-related proteins /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-234-7/.
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Heuvel, Corinna van den. "Studies on upregulation of amyloid precursor protein in response to traumatic brain injury /." Title page, contents and abstract only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phv22723.pdf.
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Ly, Philip T. T. "Glycogen synthase kinase-3 signaling in Alzheimer's disease : regulation of beta-amyloid precursor protein processing and amyloid beta protein production." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42848.
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Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that plays a part in a number of physiological processes ranging from glycogen metabolism to gene transcription. Recent studies indicated that GSK3 also involved in the formation of Alzheimer’s disease (AD) pathologies: neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles develop when abnormal tau proteins accumulate inside neurons and form insoluble filaments, and amyloid plaques develop when the amyloid β protein (Aβ) accumulates in increasingly insoluble forms. The Aβ peptide is generated through sequential cleavages of the β-amyloid precursor protein by β-secretase (BACE1) and γ-secretase. Accumulation of insoluble Aβ is believed to trigger the initial series of neurodegenerative events leading to AD. Therefore, inhibition of the pathways that lead to Aβ generation will have therapeutic implications for AD treatment.
The mechanism by which GSK3 affects APP processing and Aβ production has been controversial. Previous published reports have found differential effects on GSK3-mediated APP processing. This thesis entails a thorough investigation of GSK3’s role in APP processing and Aβ production. First, the therapeutic effects of the anti-convulsant drug, valproic acid (VPA) were tested in AD modeled mice. VPA, a known GSK3 inhibitor could interfere with Aβ production, and rescued memory deficits. In addition to inhibiting GSK3 activity, VPA also stimulate a plethora of signaling cascades. To further our understanding of GSK3’s effect on APP processing, a GSK3 specific pharmacological inhibitor (AR-A014418) and siRNA technologies were used in our systems. With specific GSK3β inhibition, we showed that BACE1-mediated cleavage of APP and Aβ production were reduced. Moreover, GSK3β induced BACE1 gene expression depends on NFκB activity. Additionally, specific inhibition of GSK3 also reduced Aβ production and neuritic plaque formation in AD modeled mice, as well as improved memory functions.
Finally, this thesis examined in detail the role of GSK3 in AD pathogenesis. This study demonstrated for the first time that the GSK3β signaling pathway regulates BACE1 transcription and facilitates Aβ production. These findings reinforced the notion that specific GSK3 inhibition is a safe and effective approach for treating AD.
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Leutz, Steffen. "Neuronaler Zelltod bei der Alzheimer-Demenz : Einfluss von b-Amyloid [Beta-Amyloid] und Amyloid-precursor-Protein /." Aachen : Shaker, 2002. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=009735379&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Nilsson, Tatjana. "Amyloid precursor protein: cellular studies and animal models /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-832-0/.
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Golde, Todd Eliot. "Analysis of the beta amyloid precursor protein mRNAs in Alzheimer's disease." Case Western Reserve University School of Graduate Studies / OhioLINK, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=case1056572599.
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Flood, Fiona. "Alzheimer's disease-related amyloid precursor protein and presenilin genes : normal function and pathophysiology /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-050-8/.
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Roberts, Hazel. "Alpha-synuclein expression influences the processing of the amyloid precursor protein." Thesis, University of Bath, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.707587.
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In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.
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Andrew, Robert. "Differential proteolysis of the amyloid precursor protein isoforms : the role of cellular location and protein-protein interactions." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/differential-proteolysis-of-the-amyloid-precursor-protein-isoforms-the-role-of-cellular-location-and-proteinprotein-interactions(5390e8fa-fc5e-4357-8109-cb2bb1c49212).html.
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Dementia, the most common cause of which is Alzheimer's disease (AD), currently affects 850,000 people in the UK, a figure set to rise to over 1 million by 2025. There is currently no disease modifying therapy available to slow or halt this progressive disease. Current understanding of AD implicates the neurotoxic amyloid-β (Aβ) peptide as the primary initiator in a cascade of events leading to the neuronal cell death and brain atrophy associated with the disease. Therefore, inhibiting the production or enhancing the clearance of Aβ within the brain has become a major target for the production of disease modifying therapeutics. Aβ is produced by brain cells through the sequential proteolytic cleavage of a larger transmembrane protein known as the amyloid precursor protein (APP) by β- and γ-secretases. Several aspects of APP physiology can influence its proteolysis, and thus Aβ production, including the isoform of APP which is expressed, its trafficking and subcellular location and its physical interactions with other proteins in the cellular environment. Here we have investigated the influence of subcellular trafficking and location and protein-protein interactions on the differential proteolysis of two APP isoforms, APP695 and APP751 in a neuroblastoma cell line. We have shown that APP751 undergoes less amyloidogenic proteolysis than APP695 and that retention within the early secretory pathway may contribute to this difference. APP751 shows higher co-localisation to the trans-Golgi network than APP695 in immunofluorescence microscopy studies, while addition of a mutation which causes APP proteolysis in the secretory pathway reduces the large difference in amyloidogenic proteolysis of these two isoforms. Targeting APP endocytosis from the cell surface, thought to be a key determinant in Aβ generation, effects APP isoform proteolysis and Aβ production to a similar extent in both the APP isoforms suggesting differences in proteolysis occur before this trafficking event. We also show by immunoblot analysis that the APP isoforms may be differentially cleaved by proteases other than β- and γ-secretase to produce recently identified proteolytic fragments. Using a liquid chromatography - tandem mass spectrometry approach coupled to prior stable isotope labelling of amino acids in cell culture (SILAC), we have identified the interactomes of the two APP isoforms in our model system. Gene ontology analysis identified enrichment of nuclear and mitochondrial proteins specifically in the APP695 interactome. Using siRNA mediated protein knockdown, we have shown interactions with Fe65 and ataxin-10 specifically influence Aβ generation from the APP695 isoform. Fe65 alters proteolysis at the rate limiting β-secretase cleavage step, while ataxin-10 alters proteolysis by γ-secretase. Interaction with growth-associated protein 43 specifically influences Aβ generation from the APP751 isoform, altering proteolysis at the γ-secretase step. Finally we have shown that recently discovered familial AD-linked mutation and protective mutation within the Aβ region of the APP protein have consistent effects on APP proteolysis in both the APP isoforms.
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Wilcock, Donna Marie. "Mechanisms of [beta]-amyloid clearance by anti-a[beta] antibody therapy." [Tampa, Fla.] : University of South Florida, 2004. http://purl.fcla.edu/fcla/etd/SFE0001169.
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Palmert, Mark Raney. "The beta amyloid protein precursor of Alzheimer's disease: Analysis of mRNAs and protein products." Case Western Reserve University School of Graduate Studies / OhioLINK, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=case1054920188.
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Lu, Daniel C. "Intracytoplasmic caspase cleavage of [beta]-amyloid precursor protein : implications for Alzheimer's disease /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3022210.
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Kirouac, Lisa. "The Concerted Regulation of Intracellular Signaling by Amyloid Precursor Protein and Aβ Peptide." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6278.
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It is widely accepted that A-beta (Aβ) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in the Alzheimer’s disease (AD) brain, yet little is known about the contribution of APP preceding AD pathogenesis. Our data presented here suggest that APP has a functional role in cell cycle regulation and proliferation. First, we demonstrat that APP is pathologically phosphorylated at Thr668 and that P-APP localizes to the centrosomes. Furthermore, P-APP is proteolytically processed in a cell cycle -dependent manner to generate its pathogenic metabolites. Using Stable Isotope Labeling by Amino Acids in Culture (SILAC) and mass spectrometry analyses, we also show that expression of APP results in the expression of proliferation-associated proteins and the phosphorylation of proteins associated with cell cycle regulation and transcription. Here, we demonstrate that APP expression and oligomeric Aβ42 elicit Ras/ERK signaling cascade and glycogen synthase kinase3 (GSK3) activation. Both ERK and GSK3 are known to induce hyperphosphorylation of tau and of APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human brain samples show increased expression of Ras, activation of GSK3 and phosphorylation of APP and tau, which correlate with Aβ levels in the AD brains. Furthermore, treatment of primary rat neurons with Aβ recapitulate these events and show enhanced Ras-ERK signaling, GSK3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Aβ induces Thr668 phosphorylation on APP, which we show enhances APP proteolysis and Aβ generation, denotes a vicious feed-forward mechanism by which APP and Aβ promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and an inhibition of this would impede the mitotic catastrophe and neurodegeneration observed in AD.
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Ostrowski, Stephen M. "Pleiotropic mechanisms of statin action in Alzheimer's Disease." Cleveland, Ohio : Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1190669698.
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Taylor, Chanel Jayne, and n/a. "Secreted amyloid precursor protein-alpha modulates hippocampal long-term potentiation, in vivo." University of Otago. Department of Psychology, 2008. http://adt.otago.ac.nz./public/adt-NZDU20081217.144344.
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Alzheimer�s disease (AD) is a neurodegenerative disorder, charaeterised by progressive loss of memory. It is important to understand what factors initiate the onset of AD so that effective therapeutic treatments can be developed to target the precise mechanisms that initiate this disease. Currently, synaptic dysfunction is widely believed to be the first significant alteration preceding the onset of AD, and is thought to be initiated by an intracellular accumulation of amyloid-β (Aβ), or a free radical-induced increase of oxidative stress. As Aβ levels rise during the onset of AD, a concomitant reduction of secreted amyloid precursor protein-α (sAPPα) is observed, as the two proteins exist in equilibrium. Intriguingly, the neuroprotective and neurotrophic properties of sAPPα indicate that it is intimately involved in the physiological pathways of the major hypotheses for the cause of AD, and may also be involved in the mechanisms that underlie learning and memory. Therefore, it is possible that during the onset of AD, the decrease of sAPPα may contribute to synaptic dysfunction by disrupting the mechanisms of synaptic plasticity.
Long-term potentiation (LTP) is the leading experimental model for investigating the neural substrate of memory formation, and describes the molecular mechanisms that underlie an increase in the strength of synaptic transmission. The role sAPPα may play in the induction and maintenance of LTP has not previously been addressed in vivo. Therefore, the aim of this thesis was to investigate whether sAPPα affects the induction of LTP in the hippocampus of the anaesthetised rat. The present findings are the first to suggest that sAPPα may modulate the induction of LTP in vivo. Decreasing the function of endogenous sAPPα (with sAPPα-binding antibodies and a pharmacological inhibition of α-secretase) significantly reduced the magnitude of LTP induced in the dentate gyrus. Therefore, the reduction of sAPPα during AD is likely to have a detrimental impact on the mechanisms of synaptic plasticity, and by extension, learning and memory. The present investigation has also found that the application of recombinant, purified sAPPα to the rat hippocampus has an �inverted U-shaped� dose-response effect on the magnitude of LTP. Low concentrations of sAPPα significantly enhanced LTP, supporting previous findings that exogenous sAPPα can facilitate in vitro LTP and enhance memory performance in animals. On the other hand, comparatively high concentrations of sAPPα significantly decreased the magnitude of LTP. This observation is also consistent with previous findings, in which high concentrations of sAPPα have been shown to be less synaptogenic and memory enhancing than lower doses. These results are the first to suggest that sAPPα modulates in vivo synaptic plasticity, and have important implications for the development of strategies to treat AD.
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Ting, Jonathan T. "Molecular mechanisms of synapse dysfunction : modeling neurological disease by viral-mediated protein overexpression in mammalian CNS neurons /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10671.
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Pasternack, Jennifer Martine. "The Alzheimer's disease beta amyloid protein precursor: Analysis of the carboxyl terminus of its soluble derivatives." Case Western Reserve University School of Graduate Studies / OhioLINK, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1060094123.
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Wiley, Jesse Carey. "Familial Alzheimer's disease mutations decrease gamma-secretase processing of beta amyloid precurson [sic] protein /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/4985.
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Rijal, Upadhaya Ajeet [Verfasser]. "Analysis of Amyloid beta (Aβ) protein in Amyloid precursor protein (APP) transgenic mouse models of Alzheimer’s disease (AD) and in human brains / Ajeet Rijal Upadhaya." Ulm : Universität Ulm. Medizinische Fakultät, 2012. http://d-nb.info/1025715012/34.
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Li, Xiaoman. "Study on memapsin 2 cleavage properties and its interacting proteins." Oklahoma City : [s.n.], 2010.
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Herber, Donna Lorraine. "Neuroinflammation in Alzheimers disease : characterization and modification of the response of transgenic mice to intrahippocampal lipopolysaccharide administration /." [Tampa, Fla.] : University of South Florida, 2004. http://purl.fcla.edu/fcla/etd/SFE0001075.
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Lebson, Lori Ann. "Monocytes as gene therapy vectors for the treatment of Alzheimer's disease." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002793.
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Zhang, Shuting. "The effect of FAD-associated mutations in amyloid-beta precursor protein and presenilin-1 genes on Alzheimer’s disease pathogenesis." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44983.
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Pathogenic mutations in amyloid-β precursor protein (APP) and presenilins (PS)
genes cause familial Alzheimer’s disease (FAD). FAD is an uncommon form of
Alzheimer’s disease (AD) with early onset (before age 65) and a rapid progression
but its neuropathology is indistinguishable from the sporadic AD. Amyloid plaque
is the unique hallmark of AD, which consists primarily of 40- and 42-residue
amyloid β protein (Aβ40 and Aβ42) with the more hydrophobic Aβ42 as its major
component. Aβ is derived from APP through sequential cleavages by β-secretase
and γ-secretase. According to the “Amyloid hypothesis”, Aβ accumulation
initiates the pathogenic cascades leading to AD, including the formation of
neurofibrillary tangles, activation of astrocytes and neuronal loss. It has been well
established that pathogenic mutations in both APP and PS genes contribute to AD
pathogenesis via impaired generation of Aβ. This powerful genetic discovery
lends great credence to the “Amyloid hypothesis”, given that APP is the precursor
of Aβ and PS acts as the enzyme to generate Aβ. The thorough understanding of
the mechanism of these pathogenic mutations could lead to decipher the AD
conundrum. Until now, all pathogenic APP mutations are autosomal dominant
mutations except for APPA673V. We discovered that APPA673V structurally
facilitates β-cleavage at Asp-1 site while inhibited the general APP processing
including all α-/β-/γ-cleavages possibly due to the intensified lysosome-dependent
degradation. The overall effect of APPA673V on the production of Aβ makes the
homozygous state necessary for APPA673V to produce enough Aβ to initiate AD
pathogenesis. Mutations in PS genes are another major cause of FAD. As another
substrate of γ-secretase apart from APP, Notch plays a fundamental role in
neurodevelopment and neurodegeneration. It has been well established that
pathogenic PS mutations impaired Notch signaling. PS1ΔS169 is a recently
discovered PS1 mutation in a Chinese FAD family. We extensively characterized
the function of PS1ΔS169 in mammalian cells and transgenic mice and found that
PS1ΔS169 promoted AD pathogenesis via altering γ-cleavage of APP without
impairing Notch processing, excluding the contribution of Notch signaling to AD
pathogenesis. Our study highlights the possibility of developing specific γ-
secretase inhibitors, which may spare Notch signaling in AD therapy.
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Vrotsos, Emmanuel George. "MCP-1 and APP involvement in glial differentiation and migration of neuroprogenitor cells." Orlando, Fla. : University of Central Florida, 2009. http://purl.fcla.edu/fcla/etd/CFE0002517.
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Wu, Jinfang. "Alzheimer's disease (AD) like pathology following developmental lead exposure in primates and the role of aging in AD-related genes regulation in rodents and primates /." View online ; access limited to URI, 2008. http://0-digitalcommons.uri.edu.helin.uri.edu/dissertations/AAI3314462.
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Xu, Guilian. "Some studies on the cholinergic and somatostatinergic systems in the brain of mouse alzheimer models with transgenes for amyloid precursor protein (APP) and presenilin." Hong Kong : University of HOng Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23540400.
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Ouellet, Stéphane. "Études des mécanismes de régulation de la transcription des gènes humains P21CIP1/WAF1, GPC3 (GLYPICAN 3) et A-beta-PP (AMYLOID beta-PRECURSOR PROTEIN)." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27537/27537.pdf.
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La connaissance des mécanismes qui contrôlent la transcription des gènes, comme l’interaction de facteurs protéiques au niveau des promoteurs, est essentielle pour comprendre plusieurs fonctions biologiques et espérer traiter certaines pathologies. Nous nous sommes attardés à mieux comprendre les mécanismes qui régulent trois gènes humains dont les patrons d’expression sont différents et qui sont impliqués dans diverses pathologies en tentant de mettre en parallèle les différences structurales et fonctionnelles entre ceux-ci. Les gènes AβPP et p21 sont exprimés chez l’adulte alors que GPC3 est exprimé uniquement chez l’embryon de façon spécifique aux tissus. L’expression d’AβPP est aussi spécifique aux tissus et sa surexpression fait partie des mécanismes mis en cause chez les patients atteints de la maladie d’Alzheimer et du syndrome de Down. Le gène p21 est quant à lui exprimé dans plusieurs types cellulaires et est fortement induit suite à des dommages à l’ADN. Enfin, nous avons montré que GPC3 est exprimé de manière différentielle dans le neuroblastome (NB) et la tumeur de Wilms (WT), deux tumeurs embryonnaires.
p21 : La caractérisation du promoteur proximal de p21 dans les fibroblastes humains normaux en prolifération nous a permis de localiser sept empreintes protéiques dont une au niveau de la séquence consensus pour NFI. Les études de retard sur gel, de transfection transitoire, d’immunoprécipitation de la chromatine et d’anti-ARN ont permis de confirmer la liaison de NFI et de le définir comme répresseur important de la transcription de p21.
AβPP : Nous avons montré que USF et Sp1 se lient au promoteur de AβPP et que leur liaison est essentielle pour générer une activité maximale du promoteur. La caractérisation in cellulo du promoteur dans les neurones et les astrocytes normaux a révélé huit sites d’interaction ADN-protéine, entre-autres au niveau des sites de liaison des facteurs de transcription CTCF, USF et Sp1.
GPC3 : La caractérisation du promoteur de GPC3 nous a permis de montrer 1) une struture chromatinienne particulière tout le long du promoteur et 2) plusieurs empreintes protéiniques putatives dont certaines spécifiques à la lignée SJNB-7 qui exprime GPC3. Parmi ces dernières, nous avons mis en évidence la liaison possible d’un facteur de transcription de type NF-Y.Gene transcription is the first step to the production of any given protein. Understanding of the molecular mechanisms regulating gene expression, such as the binding of transcription factors to genes promoters, is essential to the understanding of biological functions and to develop new powerful therapies against many clinically documented pathologies. We investigated the transcriptional regulatory mechanisms of three human genes very differently expressed and involved in diverse pathologies in an attempt to reveal structurals and functionals differencies between these mechanisms. AβPP and p21 genes are both expressed in adult while GPC3 is only transcribed in a tissus specific manner before birth. The expression of the AβPP gene is also specific to tissue and its over-expression may be involved in Alzheimer disease and Down syndrome. P21 gene is expressed in many types of cells and is strongly induced by DNA damage. Finally, we demonstrated that GPC3 is differently expressed in neuroblastoma and Wilms' tumor. P21 : The characterization of the proximal promoter from the p21 gene in normal human proliferating fibroblasts revealed seven DNA-protein footprints of which one bears a perfect consensus sequence for the NFI family of transcription factors. EMSA, CHIP, anti-RNA and transient transfection of recombinant constructs analyses clearly demonstrated that NFI interact with the most proximal LMPCR footprint on the p21 promoter and functions as a repressor. Upon serum starvation, a change in the electrophoretic mobility of the NFI DNA-protein complex was observed that may contribute to the activation mechanistic of the p21 gene throughout cell senescence and differentiation. AβPP : We demonstrated that Sp1, like USF, recognizes an element in the human AβPP gene that is necessary for full promoter activity. In cellulo footprinting analysis revealed at least eight DNA-protein interactions including CTCF, USF and many Sp1 target sites. These results were further supported by EMSA and transient transfection analysis. GPC3 : The characterisation of the entire GPC3 gene promoter revealed 1) a particular DNA structure in the promoter and 2) eight large protected regions. The use of competitor oligos in EMSA experiments and super-shift assays showed that an NFY-type transcription factor (TF) may explain the GPC3 aberrant expression in SJNB-7.
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THERIN, SEBASTIEN. "USE OF A CELL PERMEABLE PEPTIDE TO MODULATE ADAM10 SYNAPTIC LOCALIZATION AND ACTIVITY IN A MOUSE MODEL OF ALZHEIMER'S DISEASE." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/649095.
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Alzheimer’s disease (AD) is characterized by the aggregation of amyloid beta peptide (Aβ). Aβ derives from the amyloid precursor protein (APP), which can undergo two mutually exclusive pathways. The amyloidogenic pathway involves BACE and γ-secretase activities and leads to Aβ formation. While, the non-amyloidogenic pathway involves ADAM10, a disintegrin and metalloproteinase 10, which cleaves APP within the domain corresponding to Aβ, thus precluding Aβ production. Recently, we identified a new ADAM10 binding partner, named AP2, which is responsible for ADAM10 internalization, therefore affecting its activity. Interestingly, ADAM10/AP2 interaction is significantly increased in AD patients' brain compared to healthy control subjects, suggesting a role of ADAM10/AP2 in AD pathogenesis. In this framework, we have recently developed a cell permeable peptide (named PEP3) capable of interfering with ADAM10/AP2 association. The intraperitoneal administration of this CPP to a mouse model of AD for two weeks is safe and effective in impairing ADAM10 endocytosis and, thereby, in increasing ADAM10 synaptic localization. At late stages of disease, the PEP3 administration is able to change biochemical parameters, as Aβ levels and the molecular composition of the synapses without ameliorating the cognitive deficits of these mice. On the other hand, at early stage of the pathology the 14-days administration of the PEP3 rescues the cognitive impairment of the AD mice. Further investigations revealed that the synaptic levels of the NMDA receptor subunit GluN2A are increased upon the treatment and that previously observed shrinkage and dendritic spine loss in AD mice were improved after CPP treatment. These results are mediated by an increase in endogenous sAPPα. These positive results point to ADAM10 internalization as a potential target mechanism for the development of an effective AD therapy.
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Kyriazis, George A. "The endocytic protein Numb regulates APP metabolism and Notch signaling implications for Alzheimer's disease /." Orlando, Fla. : University of Central Florida, 2008. http://purl.fcla.edu/fcla/etd/CFE0002233.
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Nilsberth, Camilla. "Distribution and pathophysiological role of amyloid precursor protein and presenilin 1 : characterization in rats and in vitro studies on the pathogenic arctic mutation /." Stockholm : Karolinska Univ. Press, 2002. http://diss.kib.ki.se/2002/91-7349-329-5/.
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Jefferson, Tamara [Verfasser]. "Molecular interaction of the human metalloprotease meprin beta with the amyloid precursor protein, ADAM10, and ADAM17 based on proteomics / Tamara Jefferson." Mainz : Universitätsbibliothek Mainz, 2011. http://d-nb.info/1018183647/34.
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Fisher, Linda. "Inflammatory cytokines and NFkB in Alzheimer's disease /." Stockholm : Department of Neurochemistry, Stockholm University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-990.
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Smotherman, Jesse M. "The Impact of Causative Genes on Neuropsychological Functioning in Familial Early-Onset Alzheimer's Disease: A Meta-Analysis." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc984161/.
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Mutations of three genes encoding amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) have been shown to reliably result in familial early-onset Alzheimer's disease (FAD); a rare, but catastrophic, subtype of Alzheimer's disease (AD) marked by symptom emergence before age 65 as well as accelerated cognitive deterioration. The current study represents the first known meta-analysis on the association of APP, PSEN1 or PSEN2 on neurocognitive variables. A total of 278 FAD mutation-carriers (FAD-MC) and 284 cognitively healthy non-mutation-carriers (NC) across 10 independent investigations meeting inclusion criteria were chosen for the current meta-analysis (random effects design). Findings revealed an overarching trend of poorer performance by FAD-MC individuals compared to NC individuals across the majority of cognitive domains identified. Significant differences in effect sizes suggested FAD-MC individuals exhibited worse performance on measures of attention, explicit memory, fluency, primary memory, verbal, and visuospatial functioning. Findings indicative of differential sensitivity to cognitive domain impairments across FAD-MC and NC groups inform neuropsychological descriptions of individuals in preclinical phases of FAD.
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Linning, Philipp, Ute Haussmann, Isaak Beyer, Sebastian Weidlich, Heinke Schieb, Jens Wiltfang, Hans-Wolfgang Klafki, and Hans-Joachim Knölker. "Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores – development of potential agents for the treatment of Alzheimer's disease." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-138993.
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Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP)Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Itkin, Anna. "Multidisniplinary study of Alzheimer's disease-related peptides : from amyloid precursor protein (APP) to amyloid β-oligomers and γ-secretase modulators." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAF051/document.
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Une des caractéristiques histopathologiques de la maladie d'Alzheimer (AD) est la présence de plaques amyloïdes formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de l'APP. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, les peptides APP_TM4K ont été étudiés dans la bicouche lipidique en utilisant l’ATR-FTIR et ssNMR. Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, hétérogénéité de conformation et d'orientation a été observée pour le site de clivage γ et , que peuvent avoir des implications dans le mécanisme de clivage et donc dans la production d’Aβ. Les peptides Aβ s'agrègent pour produire des fibrilles et aussi de manière transitoire d'oligomères neurotoxiques. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. Ces résultats nous ont amené à conclure que les Ca2+ stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans l’AD. Les positions et une orientation précise de deux nouveaux médicaments puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen dans la bicouche lipidique, ont été obtenus à partir des expériences des ssNMR. Ces résultats indiquent que le mécanisme probable de modulation du clivage par la y-sécrétase est une interaction directe avec le domaine TM de l’APPA histopathological characteristic of Alzheimer’s disease (AD) is the presence of amyloid plaques formed by amyloid β(A) peptides of 40 and 42 residues-long, which are the cleavage products of APP by proteases. To understand the role of structural changes in the TM domain of APP, APP_TM4K peptides were studied in the lipid bilayer using ATR-FTIR and ssNMR. While the overall secondary structure of the APP_TM4K peptide is helical, conformational and orientational heterogeneity was observed for the y- and for the -cleavage sites, which may have implications for the cleavage mechanism and therefore the production of Aβ. Starting from its monomeric form, Aβ peptides aggregate into fibrils and / or oligomers, the latter being the most neurotoxic. We found that in the presence of Ca2 +, Aβ (1-40) preferably forms oligomers, whereas in the absence of a2 + Aβ (1-40) aggregates into fibrils. In samples without Ca2 +, ATR-FTIR shows conversion from antiparallel β sheet conformation of oligomers into parallel β sheets, characteristic of fibrils. These results led us to conclude that Ca2 +stimulates the formation of oligomers of Aβ (1-40), that have been implicated in the pathogenesis of AD. Position and precise orientation of two new drugs powerful modulators of γ-secretase benzyl-carprofen and carprofen sulfonyl in the lipid bilayer were obtained from neutron scattering and ssNMR experiments. These results indicate that carprofen-derivatives can directly interact with APP. Such interaction would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by β -secretase, diminishing or greatly reducing Aβ42 production
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Linning, Philipp, Ute Haussmann, Isaak Beyer, Sebastian Weidlich, Heinke Schieb, Jens Wiltfang, Hans-Wolfgang Klafki, and Hans-Joachim Knölker. "Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores – development of potential agents for the treatment of Alzheimer's disease." Royal Society of Chemistry, 2012. https://tud.qucosa.de/id/qucosa%3A27800.
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Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP).Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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許瑰蓮 and Guilian Xu. "Some studies on the cholinergic and somatostatinergic systems in the brain of mouse alzheimer models with transgenes for amyloid precursorprotein (APP) and presenilin." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31242534.
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Ill-Raga, Gerard. "Study of the pathophysiological role of nitric oxide and nitrative stress in brain: translational effects on the cleavage of the amyloid precursor protein in Alzheimer's disease and post-translational effects on fibrinogen in brain ischemia." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/31907.
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Nitric oxide (NO) is a neurotransmitter involved in memory processes. Currently, the
only recognized physiological signalling pathway controlled by NO is the activation of
guanylyl cyclase. In this thesis, we propose an alternative NO-signalling pathway that
involves the Heme-regulated eukaryotic initiation factor-2a kinase (HRI) and eIF2a
phosphorylation. We have found that the enzyme BACE1, a key protein in Alzheimer’s
disease (AD), is controlled by this novel pathway. This pathway would be involved in
the physiology of memory formation and learning processes. We have also studied how
an external stress factor, the Herpes Simplex Virus 1, can disrupt this cascade leading to
a pathological increase in BACE1 and amyloid ß-peptide (Aß) production. Aß
aggregates forming fibrils that generate free radicals. These react with NO producing
peroxynitrite, which contribute to AD progression. Since NO turns toxic when produced
in a pro-oxidant environment we have also studied the effect of peroxynitrite in Stroke.L’òxid nítric (NO) és un neurotransmissor involucrat en processos de memòria. Actualment, l’única cascada de senyalització fisiològica controlada per NO consisteix en l’activació de la guanilat ciclasa. En aquesta tesi, en proposem una d’alternativa que inclou la fosforilació de eIF2a per la Heme-regulated eukaryotic initiation factor-2a kinase (HRI). Hem mostrat com l’enzim BACE1, una proteïna clau en la malaltia d’Alzheimer (AD), és controlat per aquesta nova cascada de senyalització, que podria estar involucrada en la fisiologia de l’aprenentatge i la memòria. També hem estudiat com un factor d’estrès extern, l’ Herpes Simplex Virus 1, pot pertorbar aquesta cascada donant lloc a increments patològics en BACE1 i pèptid ß-amiloide (Aß). L’Aß agrega formant fibril·les que generen radicals lliures. Aquests reaccionen químicament amb NO produint peroxinitrit, que contribueix a la progressió de l’AD. Pel fet que l’NO esdevé tòxic quan és produït en un entorn pro-oxidant, hem estudiat també l’impacte que el peroxinitrit té en l’ictus.
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MAROLDA, ROBERTA. "Effetto antiamiloidogenico della Sostanza P in un modello apoptotico di granuli cerebellari: possibili implicazioni nella patologia di Alzheimer." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/209099.
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La Sostanza P (SP) è un neuropeptide di 11 aminoacidi, membro della famiglia delle tachichinine, ampiamente distribuita nel sistema nervoso centrale ed ha un ruolo funzionale sia in condizioni fisiologiche che patologiche, tra le quali le malattie neurodegenerative (Raffa, 1998; Severini et al., 2002). Alterati livelli di SP sono stati riscontrati nelle regioni corticali di tessuti cerebrali di pazienti malati di Alzheimer (AD) (Quigley and Kowall, 1991; Waters and Davis, 1997). Recentemente, è anche stata riportata una riduzione della SP nella corteccia cerebrale, nell’ippocampo, nei gangli basali e nel fluido cerebrospinale dei pazienti malati di AD, il che indica un possibile ruolo svolto dalla SP in questa patologia (Jiménez-Corral et al., 2006). Sulla base di numerose evidenze in vitro e in vivo che dimostrano il ruolo neuroprotettivo della SP, abbiamo utilizzato un modello di granuli cerebellari di ratto in vitro (CGCs). In condizioni di basse concentrazioni di K+ questi neuroni vanno incontro a morte neuronale per apoptosi con attivazione del processamento amiloidogenico dell’APP, mimando così i meccanismi molecolari che avvengono in vivo nella patologia di AD. Dati recenti hanno evidenziato che sostanze in grado di stimolare gli enzimi dell’α-secretasi (ADAM) e quindi la promozione della via non amiloidogenica dell’APP potrebbero avere una potenziale attività terapeutica nella patologia. Scopo del presente lavoro è stato quello di valutare il possibile effetto della SP sul processamento proteolitico dell’APP, con particolare interesse verso la via dell’α-secretasi. Abbiamo trovato che la SP, ad una concentrazione di 200nM, protegge i CGCs dalla morte per apoptosi, riduce la produzione di strutture β extracellulari e i livelli di Aβ1-42 intracellulari. Inoltre abbiamo dimostrato che la SP induce il processamento non amiloidogenico dell’APP, promuovendo la secrezione del frammento neuroprotettivo solubile APPα, aumenta l’attività enzimatica dell’α-secretasi, l’espressione genica e proteica di ADAM 9 e la maturazione di ADAM 10, senza influenzare l’espressione del precursore proteico dell’Aβ (APP) e di BACE 1, l’enzima coinvolto nel processamento amilodogenico dell’APP.
In conclusione, il presente studio mette in evidenza che la SP, attivando la via non amilodogenica, potrebbe assumere un’importanza clinica come agente in grado di modificare la patologia di AD.Substance P (SP) is an 11-aa neuropeptide, member of the tachykinins (TK) family, broadly distributed in the central nervous system and having a functional role both in physiological and pathological conditions, as in neurodegenerative diseases (Raffa, 1998, Severini et al., 2002). Altered levels of SP have been observed in the cortical regions of post-mortem brain tissues from patients with Alzheimer’s disease (AD) (Quigley and Kowall, 1991; Waters and Davis, 1997). Recently, a consistent SP reduction in the cerebral cortex, hippocampus, basal ganglia and cerebrospinal fluid of AD patients was reported, indicating a possible role of SP in the progression of this disease. On the basis of the in vitro and in vivo results demonstrating the involvement of SP in neuroprotection, we used CGCs in low K+ conditions. In this model, CGCs undergo to apoptotic cell death with activation of amyloidogenic processing of APP, mimicking molecular mechanisms that occur in vivo in AD. Recent data demonstrated that drugs that can regulate the processing of APP towards the non-amyloidogenic pathway (ADAM) may have a therapeutic potential in AD. Aim of the present work was to assess the possible effects of SP on proteolytic processing of APP, with particular interest towards the α-secretase pathway. Data of the present work demonstrate that SP, at a concentration of 200nM, protects CGCs against apoptotic cell death induced by low K+ conditions, significantly reduces the extracellular fibrils production and levels of intracellular Aβ1-42. In addiction, we demonstrate that SP increases α-secretase activity, through the secretion of the neuroprotective soluble fragment APPα, induces the activation of α-secretase enzymatic activity, ADAM 9 gene and protein expression, ADAM 10 maturation, without influencing the precursor protein expression of Aβ (APP) and of BACE 1, the enzyme involved in the amylodogenic processing of APP. In conclusion, this study demonstrates that SP, by activating the non-amyloidogenic processing of APP, may have a therapeutic potential as disease-modifying agent in AD.
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Arano, Rodriguez Ivan. "Rab Proteins and Alzheimer's: A Current Review of Their Involvement in Amyloid Beta Generation with Focus on Rab10 Expression in N2A-695 Cells." BYU ScholarsArchive, 2015. https://scholarsarchive.byu.edu/etd/5648.
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This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.
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Mora, García Natalia. "Analisis de la expresión y la función del gen beta-amyloid protein precursor like en relación a la vía de RasI en el disco imaginal de ojo de Drosophila melanogaster." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/104149.
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Drosophila es un modelo versátil para entender las bases genéticas de la señalización celular. En particular, el ojo compuesto de Drosophila proporciona un tejido ideal para estudiar los mecanismos de integración de señales que dirigen la formación de la red de neuronas fotorreceptoras. La vía de Ras/MAPK está involucrada en la determinación de todos los fotorreceptores, pero es particularmente necesaria para la especificación del fotorreceptor R7, ya que su ausencia determina la transformación del R7 en una célula no neural. De manera que durante el desarrollo del ojo, Ras media la decisión entre la diferenciación neural y no neural. Sin embargo, se desconoce el perfil de genes activados por Ras. Con el objetivo de describir este perfil, hemos realizado microarrays comparando diferentes alelos del receptor tirosina quinasa Sevenless (Sev) que participa en la especificación del R7 mediante la activación de la vía de Ras / MAPK. Uno de los genes que responden a la activación de Sev es el gen amyloid protein precursor-like (Appl). El incremento de la expresión de Appl detectado en los microarrays, también se confirmó por hibridación in situ.
En un estudio detallado de localización de la proteína, observamos que aunque está presente en todos los fotorreceptores, hay más proteína Appl en R7 y R8, que en los otros. Para evaluar si Ras es necesario para la activación de Appl, realizamos clones con el alelo dominante negativo del gen Epithermal Growth factor Receptor (DERDN). En estos clones no se detectó Appl, mientras que los clones del alelo constitutivamente activo de Ras; RasV12 dieron lugar a la sobreexpresión de Appl. En cualquier otro tejido, RasV12 no produjo expresión ectópica, indicando que Ras es necesario y suficiente para la expresión de Appl aunque esta regulación es dependiente del dominio del ojo. Para ver si el factor de transcripción de la vía; Pnt, es capaz de unirse a Appl para activar su expresión realizamos clones con el alelo de pérdida de función pntΔ88. Estos clones mostraron que la falta de pnt tiene como resultado la ausencia de Appl. En segundo lugar evaluamos la posible unión directa de Pnt a Appl mediante transgénicos de regiones supuestamente enhancers de Appl unido a un promotor mínimo y al gen reportero lacZ. Ninguna de las construcciones fue capaz de dirigir la expresión de lacZ en ojo, sin embargo dos de ellas fueron capaces de dirigirla en cerebro, sugiriendo que estas dos secuencias actúan como unidades reguladoras de la expresión de Appl. Ya que no produjeron expresión en ojo, decidimos sensibilizar la respuesta de las construcciones a Ras, incrementando la actividad de la vía mediante clones RasV12. En este nuevo contexto, los dos ETS que tenían expresión en cerebro, fueron capaces de dirigir la expresión de lacZ. Además la unión de pnt a Appl se confirmó mediante experimentos de InmunoPrecipitación de la Cromatina (CHIP).
Ras en ojo es responsable de la determinación de casi todos los fotorreceptores, sin embargo observamos que mientras que la determinación no está afectada en mutantes Appld, sí lo está el funcionamiento del R7. Este fotorreceptor es el único capaz de ver la luz ultravioleta. Mediante experimentos de comportamiento observamos que las moscas Appld tienen disminuida la capacidad de discernir la luz UV. Esta reducción es debida en parte porque el 2% de los axones de R7 de moscas Appld no llegan a hacer la sinapsis. Para tratar de incrementar los efectos observados y poder describir con mayor claridad la función de Appl en ojo, testamos la habilidad de los mutantes Appld de discernir el UV, en combinación con mutantes heterocigotos de proteínas descritas en el proceso de guía de axones. La perdida de función de Appl combinada con el mutante heterocigoto del gen neurotactina (nrt), produjo un claro deterioro de la capacidad del R7 para discernir UV y de los axones pare llegar a hacer la sinapsis demostrando que Appl es necesario para la correcta función del R7.In a genome wide expression profile search for genes that characterize the Drosophila R7 photoreceptor specification we found Appl, the ortholog of human APP and a key factor in the pathogenesis of Alzheimer’s disease. We analyzed Appl expression in the eye imaginal disc and found that is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, we have analyzed Appl null mutants and found reduced preference for UV light, likely due to mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK activated by the tyrosine kinase receptor Sevenless. Thus, we have studied whether Ras1/MAPK is responsible for the high Appl expression. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. We show that Appl is directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Also, the zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism.
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Pavoni, Serena. "Mise au point d’un nouveau modèle d’organoïde cérébral humain pour l’étude des mécanismes d’interaction de la protéine prion et de l’amyloïde β." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS427.
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Les mécanismes de type prion sont désormais reconnus comme sous-tendant la plupart des maladies neurodégénératives humaines, avec en premier lieu la maladie d’Alzheimer (MA) au niveau de ses 2 marqueurs spécifiques, l’amyloïde β (Aβ à l’origine de l’hypothèse étiopathogénique de la cascade amyloïde) et la protéine Tau phosphorylée. Par ailleurs la protéine du prion (PrPC) est décrite comme interagissant à de multiples niveaux avec le métabolisme de l’Aβ sans que les mécanismes physiopathologiques sous-jacents n’aient pu être expliqués. Pour sortir de l’impasse actuelle concernant le développement d’approches thérapeutiques efficaces pour la MA, l’industrie pharmaceutique a besoin de modèles expérimentaux innovants. En effet, à ce jour aucun modèle in vivo, en dépit des progrès réalisés avec les souris transgéniques, n’arrive à refléter la complexité cérébrale humaine ni à mimer une MA clinique. Les cultures in vitro en 2D sont quant à elles très éloignées des situations conduisant à l’accumulation d’agrégats protéiques pathologiques. Le but de notre thèse a été d’utiliser dans le domaine des neurosciences les nouvelles perspectives de recherche ouvertes par les technologies des cellules souches pluripotentes induites (cellules iPS) en développant un modèle de différentiation en 3D pour obtenir des organoïdes cérébraux humains (OC) (mini cerveaux). Leur capacité d’auto-organisation en 3D de tissu neuroectodermique nous a permis de recréer un système complexe mimant différentes structures cérébrales humaines dans lesquelles nous avons pu caractériser les marqueurs attendus. L’étude de l’expression des protéines d’intérêt APP et PrPC pendant la différentiation neurale a permis de caractériser la modulation des niveaux des deux protéines en fonction du temps de culture. Afin d’orienter le modèle vers des mécanismes d’accumulation protéique de type MA, nous avons testé différents inducteurs chimiques dont l’Aftin-5 qui est capable de moduler les voies post-traductionnelles de l’APP. Plusieurs stratégies de traitement ont été adoptées pour induire le clivage de l’APP et la génération d’Aβ. La production des fragments solubles Aβ38, Aβ40, Aβ42 a été mise en évidence par ELISA. Les niveaux générés sont reproductibles et l’augmentation du ratio Aβ42/Aβ40 est cohérente avec les données extrapolées des modèles murins et humains, ce qui a permis de valider notre modèle. Les niveaux d’expression génique et protéique de PrPC et de APP suite au traitement ont été analysés afin de mieux déterminer le rôle de l’interaction entre ces deux facteurs. L’objectif à long terme consiste à améliorer ce modèle, dont les limites actuelles sont notamment l’absence de vascularisation et le niveau de maturation du tissu neural. Le défi majeur dans le cadre de la culture des OC consiste donc à favoriser l’intégration du système vasculaire, et par ailleurs à accélérer le vieillissement in vitro pour l’étude de maladies neurodégénératives. La perspective de pouvoir automatiser le système de culture des OC permet d’envisager l’utilisation de ce modèle à plus grande échelle dans le cadre de test de cytotoxicité et/ou de criblage pharmacologique à haut débit pour identifier de nouvelles molécules thérapeutiques pour la MAPrion-like mechanisms are known to underlie most of human neurodegenerative diseases including Alzheimer’s disease (AD), which is characterized by two important pathological markers, β amyloid (or Aβ at the origin of the etiopathogenic amyloid cascade hypothesis) and phosphorylated tau protein. Furthermore, the prion protein (PrPC) interacts at multiple levels with the metabolism of Aβ, by mechanisms which are not well understood. To overcome the current limits in the development of efficient strategies to treat AD, the pharmaceutical industry requires innovative experimental models. However, even if a lot of progress has been achieved by using transgenic mouse models, to date no in vivo model can reflect the complexity of human brain or reproduce a clinical context. 2D in vitro cell culture models are unable to allow the aggregation and accumulation of pathological proteins as observed in vivo. The aim of this study consists in taking advantage of the research prospects offered by induced pluripotent stem cell (iPSCs) in the field of neurosciences. iPSCs can be used to generate 3D models of differentiation also called human cerebral organoids or mini-brains (MBs). Their ability to self-organise in 3D neuroectodermic tissue leds to a complex system that mimics different human cerebral structures in which we were able to characterize the expected markers. The study of the two proteins of interest (APP and PrPC) during neural differentiation has allowed us to follow the modulation of protein expression level occurring during the in vitro development of the human MBs. In order to use this model to reproduce the protein accumulation mechanisms seen in AD, we have tested chemical inductors such as Aftin-5 in order to modulate the APP post-transcriptional pathway towards a pathological outcome. Many strategies of treatment are adopted to lead APP cleavage and Aβ generation. The production of soluble fragments Aβ38, Aβ40, Aβ42 in the supernatant of organoids has been showed using ELISA technique. The levels generated are reproducible and the increase of Aβ42/Aβ40 ratio is consistent with extrapolated data from mouse and human models thus validating our model. Analysis at the gene and protein level has been assessed in order to understand the interaction between PrPC and APP after treatment. The long-term goal consists in improving this model which is notably hampered by the absence of vascularization and the low level of maturation of the neural tissue. The main challenge in MB culture thus consists in the integration of the vascular system, and also in increasing the speed of ageing process in vitro for the study of neurodegenerative diseases. In the long term, the prospect of automating the culture of MBs would allow the use of the system for cytotoxicity testing and/or high throughput screening for the discovery of new drugs for AD
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Fiorelli, Tina N. "Proteolytic Processing of the Amyloid Precursor Protein During Apoptosis and Cell Cycle: Implications for Alzheimer's Disease." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4486.
Full textAbstract:
Alzheimer's disease is characterized by the presence of amyloid plaques, made up primarily of Aϐ peptides, and neurofibrillary tangles, containing hyperphosphorylated tau. Aϐ is generated by sequential proteolysis of the amyloid precursor protein (APP) by beta and gamma secretases. The leading hypothesis of Alzheimer's disease pathogenesis is the amyloid cascade hypothesis, which suggests that amyloid is central to the disease process. However, tau pathology correlates more closely with cognitive dysfunction and follows a predictable anatomical course through the brain. We hypothesize that if Aϐ is upstream of tau pathology and tau pathology follows this predictable course through the brain, Aϐ production may also propagate through the brain in an anatomical fashion. In order to investigate this possibility, we examined two broad cellular processes induced in cells when exposed to Aϐ, p53-dependent apoptosis and cell cycle activation. We report that p53-dependent apoptosis is associated with a decrease in the Aϐ and sAPP-alpha and an increase in an alternative, caspase-cleaved fragment of APP, resulting from an apparent cleavage in the near extracellular domain of APP. Mitosis is associated with the phosphorylation of both tau and APP, and increased production of Aϐ. Our results indicate that while p53-dependent apoptosis is not associated with increased amyloidogenesis, cell cycle activation increases Aϐ production and may play a role in disease propagation. Together, these findings suggest various treatment approaches, including cell cycle inhibition and disruption of APP endocytosis, which may decrease amyloidogenic processing. Continued research into these potential approaches, coupled with earlier detection of the disease process, could lead to promising treatments for Alzheimer's disease.
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Guix, Ràfols Francesc Xavier. "Study of the pathophysiological role of nitric oxide on the amyloid-induced toxicity attending to the biochemical modifications and cellular damages." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7162.
Full textAbstract:
Aquesta tesi demostra que el peroxinitrit produït com a conseqüència del pèptid beta-amiloide (A) contribueix l'augment de la relació A42/A40 que ocorre a la malaltia d'Alzheimer. L'A42 contribueix a l'aparició de la malaltia degut a la seva major toxicitat (quan es compara amb l'A40) que resulta d'una gran estabilitat i capacitat agregativa. A més el peroxinitrit incrementa la toxicitat d'aquest degut a què potencia la seva agregació en forma d'oligomers altament tòxics. De fet els oligomers formats de nitro-A42 presenten una major toxicitat que aquells formats de A42 . En conjunt aquest resultats senyalen l'important paper que l'A42 té en la malaltia d'Alzheimer.Per altra banda, des de la identificació dels agregats d'A i la subseqüent formació dels cabdells neurofibrilars (NFT) com a els dos trets distintius de la malaltia, un gran esforç s'ha dedicat a establir els mecanismes moleculars que uneixen ambdós processos. Aquesta tesi demostra que el peroxinitrit format a partir de l'agregació de d'Ai la conseqüent nitrotirosinació de proteïnes, potencia l'agregació de la proteïna tau en forma de fibres. D'aquesta forma, la nitrotirosinació de la proteïna triosafosfat isomerasa (TPI) podria ser el vincle entre la toxicitat derivada del agregats d'Ai la patologia derivada de la proteïna tau. Per tant, la nitrotirosinació de la TPI podria explicar la progressió temporal que ocorre als cervells de pacients amb la malaltia d'Alzheimer des de la toxicitat induïda per l'Ai l'aparició dels NFT. Els resultats presentats en aquesta tesi podrien obrir nous aspectes en la recerca de la malaltia d'Alzheimer així com en altres malalties que cursin amb estrès oxidatiu i plegament erroni de proteïnes.
This thesis demonstrates that amyloid ß-peptide (Aß)-induced peroxynitrite contributes to the switch of the Aβ42/Aβ40 ratio that occurs in Alzheimer's disease (AD). Since Aβ42 is more toxic due to its higher aggregation and stability, it contributes to the trigger of the disease. In addition the aggregation of Aβ42 in form of the highly toxic oligomers is incremented by the presence of peroxynitrite. Moreover, these nitro-Aß42 oligomers are more toxic than those non-nitrated. All these results support the important role of peroxynitrite in AD etiology.
Furthermore, since the identification of Aß accumulation and the subsequent formation of neurofibrillary tangles (NFT) as the two defining pathological hallmarks of AD, a fair amount of research on AD has been driven by the need to find the molecular mechanism linking Aß and NFT. This thesis shows the Aß-induced peroxynitrite, and the consequent nitrotyrosination of proteins, promotes tau fibrillization. Thus triosephosphate isomerase (TPI) nitrotyrosination could be the link between Aß-induced toxicity and tau pathology. Therefore, TPI nitrotyrosination may explain the temporal progression from Aß toxicity to NFT formation in AD brain. The work presented in this thesis could open a novel angle in the research of the pathophysiology of AD and could also have an impact to the research in other neurodegenerative diseases involving oxidative stress and protein misfolding.