| 1. |
- Hedskog, Louise, et al.
(författare)
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Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:19, s. 7916-7921
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Tidskriftsartikel (refereegranskat)abstract
- It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and sigma 1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)(Swe/Lon) mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid beta-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.
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| 2. |
- Larssen, Pia
(författare)
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Advances in exosome-mediated immunotherapy and diagnostics
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Exosomes are small vesicles with immune-stimulatory capacity, which can activate T cell responses in a B cell dependent manner, and therefore may serve as immune therapeutic tools. Peptide-loaded dendritic cell (DC)-derived exosomes are proven safe in clinical trials, although with limited ability to induce cytotoxic T lymphocyte (CTL) responses or prolong patient survival. Therefore, we aimed to investigate the role of exosomal MHC/peptide complexes in immune activation and explore how to enhance exosome induced immunotherapies by applying additional stimuli to the exosomes. Bone marrow-derived dendritic cell (BMDC) exosomes loaded with ovalbumin (OVA) and α-galactosylceramide (αGC) were used for this purpose. Exosomes lacking major histocompatibility complex (MHC) class I or those that were MHC mismatched were thoroughly studied in vivo for their ability to stimulate effector T cells and humoral responses. In addition, we applied a novel strategy, lyophilization, for exosomal loading of antigen and adjuvants. Here, OVA, CpG-ODN and αGC were added to RAW 264.7-derived exosomes and assessed for their immune-stimulatory capacity. We demonstrated that exosomal MHC/peptide complexes were redundant for T cell stimulation in vivo in the presence of whole OVA, as MHCI-/- and allogeneic exosomes could successfully induce CD8+ T cell responses and inhibit tumor progression (study I). Importantly, allogeneic exosomes served as an adjuvant by the upregulation of T follicular helper (Tfh) cells and increased antigen-specific antibody production (study II). We also discovered that lyophilization was feasible for loading exosomes without markedly altering exosome characteristics. Notably, additional use of the TLR9 ligand CpG-ODN improved their immune-stimulatory properties and achieved tumor regression (study III). Selective loading and accumulation of certain tissue-specific proteins and RNA into exosomes provides a platform for potential biomarker analysis, the advantages of which include the accessibility of vesicles in body fluids (“liquid biopsies”), and the ability to trace cellular origin. However, limited material often restricts exosome proteomic analyses. Therefore, we aimed at applying the highly sensitive proximity extension assay (PEA) on cell line- and body fluid-derived exosomes to investigate the potential of using PEA for exosome protein evaluation. We confirmed that PEA can be applied on exosomes to trace their cellular source and to identify accumulated vesicle proteins. Also, the protein content of the body fluid-derived exosomes from breast milk and seminal fluid displayed diverse protein profiles (study IV), suggesting the cell/tissue traceability of exosomes by PEA and motivating their future use as biomarkers. In conclusion, this thesis provides increased understanding of the mechanisms underlying exosome-based immunotherapies and suggests the use of impersonalized exosomes and allogenicity as a possible means of enhancing their immune-stimulatory effects in a clinical setting. In addition, this thesis offers insight into novel technologies for improved exosomal loading and the use of PEA for exosome proteomic research.
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| 3. |
- Larssen, Pia, et al.
(författare)
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Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay
- 2017
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 16:3, s. 502-511
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.
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| 4. |
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| 5. |
- Weber, Tobias A, et al.
(författare)
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γ-Secretase modulators show selectivity for γ-secretase-mediated amyloid precursor protein intramembrane processing.
- 2022
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Ingår i: Journal of cellular and molecular medicine. - : Wiley. - 1582-4934 .- 1582-1838. ; 26:3, s. 880-892
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of β-amyloid peptide 42 results in the formation of toxic oligomers and plaques, which plays a pivotal role in Alzheimer's disease pathogenesis. Aβ42 is one of several Aβ peptides, all of Aβ30 to Aβ43 that are produced as a result of γ-secretase-mediated regulated intramembrane proteolysis of the amyloid precursor protein. γ-Secretase modulators (GSMs) represent a promising class of Aβ42-lowering anti-amyloidogenic compounds for the treatment of AD. Gamma-secretase modulators change the relative proportion of secreted Aβ peptides, while sparing the γ-secretase-mediated processing event resulting in the release of the cytoplasmic APP intracellular domain. In this study, we have characterized how GSMs affect the γ-secretase cleavage of three γ-secretase substrates, E-cadherin, ephrin type A receptor 4 (EphA4) and ephrin type B receptor 2 (EphB2), which all are implicated in important contexts of cell signalling. By using a reporter gene assay, we demonstrate that the γ-secretase-dependent generation of EphA4 and EphB2 intracellular domains is unaffected by GSMs. We also show that γ-secretase processing of EphA4 and EphB2 results in the release of several Aβ-like peptides, but that only the production of Aβ-like proteins from EphA4 is modulated by GSMs, but with an order of magnitude lower potency as compared to Aβ modulation. Collectively, these results suggest that GSMs are selective for γ-secretase-mediated Aβ production.
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