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Elevated MARK2-Dependent Phosphorylation of Tau in Alzheimer's Disease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. (Prof. Ulf Landegren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. (Prof. Ulf Landegren)
Dept. of Neuroscience, iMed, CNS and Pain Södertälje, AstraZeneca Research and Development.
Dept. of Neuroscience, iMed, CNS and Pain Södertälje, AstraZeneca Research and Development.
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2013 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 33, no 3, 699-713 p.Article in journal (Refereed) [Artistic work] Published
Abstract [en]

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

Place, publisher, year, edition, pages
2013. Vol. 33, no 3, 699-713 p.
Keyword [en]
Alzheimer's disease, MARK, phosphorylation, proximity ligation assay, tau
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-179823DOI: 10.3233/JAD-2012-121357ISI: 000313620500005OAI: oai:DiVA.org:uu-179823DiVA: diva2:546476
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Proximity Ligation Assay for High Performance Protein Analysis in Medicine
Open this publication in new window or tab >>Proximity Ligation Assay for High Performance Protein Analysis in Medicine
2012 (English)Doctoral thesis, comprehensive summary (Other academic) [Artistic work]
Abstract [en]

High quality reagents are preconditions for high performance protein analyses. But despite progress in some techniques, e.g. mass spectrometry, there is still a lack of affinity-based detection techniques with enhanced precision, specificity, and sensitivity. Building on the concept of multiple affinity recognition reactions and signal amplification, a proximity ligation assay (PLA) was developed as a molecular tool for analyzing proteins and their post-translational modification and interactions. PLA enhanced the analysis of protein expression levels and post-translational modifications in western blotting (Paper I), which had elevated sensitivity and specificity, and an ability to investigate protein phosphorylation.

A general and straightforward method was established for the functionalization of affinity reagents through adding DNA strands to protein domains for protein analysis in medicine (Paper II). A method for protein domain-mediated conjugation was developed to simplify the use of recombinant affinity reagents, such as designed ankyrin repeat protein (DARPin), in DNA-mediated protein analyses.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline and memory impairment, and amyloid-beta plaques and neurofibrillary tangles (NFT) in the brain are clinical hallmarks of the disease. In order to understand the mechanisms underlying the formation of NFT, in situ PLA was used to explore the role of microtubule affinity related kinase 2 (MARK2) in phosphorylating tau protein during the pathological progress of AD (Paper III). The analyses of roles of MARK proteins 1-4 in phosphorylating tau protein in cells and in post-mortem human brains were performed in Paper IV.

The focus of this thesis was the study of post-translational modifications and interactions of proteins in medicine. Procedures for high performance protein analysis in western blotting via proximity ligation were developed, and a functionalization method for recombinant affinity reagents in DNA-mediated protein analysis was established. These and other techniques were used to investigate the roles of tau-phosphorylating MARK family proteins in AD.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 801
Keyword
protein, post-translational modification, interactions, protein domain, western blotting, MARK, tau, AD
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-179827 (URN)978-91-554-8449-1 (ISBN)
Public defence
2012-10-05, Rudbeck hall, Rudbeck Laboratory, Dag Hammarskjölds väg20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-08-23 Last updated: 2013-01-22Bibliographically approved
2. Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
Open this publication in new window or tab >>Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed.

In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences.

In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA).

In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls.

In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS.

In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 980
Keyword
proximity ligation assay, proximity barcoding assay, sensitive, in situ, multiplex, profiling, amyloid-beta, MARK, tau, CSF, biomarker, protein complexes, Alzheimer’s disease, amyotrophic lateral sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-220070 (URN)978-91-554-8901-4 (ISBN)
Public defence
2014-04-25, B21, Biomedicinskt centrum, Husargatan 3, SE-75123, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-04-03 Created: 2014-03-10 Last updated: 2014-04-29

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Gu, Gucci JijuanWu, DiNilsson, LarsLandegren, UlfKamali‐Moghaddam, Masood

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