| 1. |
- Ansell - Schultz, Anna, et al.
(författare)
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Reduced retromer function results in the accumulation of amyloid-beta oligomers
- 2018
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Ingår i: Molecular and Cellular Neuroscience. - : Academic Press. - 1044-7431 .- 1095-9327. ; 93, s. 18-26
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimers disease (AD) is a neurodegenerative disorder characterized by a progressive loss of multiple cognitive functions. Accumulation of amyloid beta oligomers (oA beta) play a major role in the neurotoxicity associated with the disease process. One of the early affected brain regions is the hippocampus, wherein a reduction of the vacuolar protein sorting-associated protein 35 (VPS35), the core protein comprising the retromer complex involved in cellular cargo sorting, has been identified. To investigate the role of the retromer function on the accumulation and clearance of oA beta, we reduced retromer function by selectively inhibiting VPS35 gene expression using siRNA in differentiated neuronal SH-SY5Y cells. As cell-to-cell transfer of oA beta to new brain regions is believed to be important for disease progression we investigated the effect of VPS35 reduction both in cells with direct uptake of oA beta and in cells receiving oA beta from donor cells. We demonstrate that reduced retromer function increases oA beta accumulation in both cell systems, both the number of cells containing intracellular oA beta and the amount within them. This effect was shown at different time points and regardless if the AD originated from the extracellular milieu or via a direct neuronal cell-to-cell transfer. Interestingly, not only did reduced VPS35 cause oA beta accumulation, but oA beta treatment alone also lead to a reduction of VPS35 protein content. The accumulated oA beta seems to co-localize with VPS35 and early endosome markers. Together, these findings provide evidence that reduced retromer function decreases the ability for neurons to transport and clear neurotoxic oA beta received through different routes resulting in the accumulation of oA beta. Thus, enhancing retromer function may be a potential therapeutic strategy to slow down the pathophysiology associated with the progression of AD.
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| 2. |
- Sackmann, Valerie, et al.
(författare)
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Anti-inflammatory (M2) macrophage media reduce transmission of oligomeric amyloid beta in differentiated SH-SY5Y cells
- 2017
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Ingår i: Neurobiology of Aging. - : ELSEVIER SCIENCE INC. - 0197-4580 .- 1558-1497. ; 60, s. 173-182
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Tidskriftsartikel (refereegranskat)abstract
- Neuroinflammation plays an influential role in Alzheimers disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oA beta) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oA beta, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oA beta and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies. (C) 2017 Elsevier Inc. All rights reserved.
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| 3. |
- Sackmann, Valerie, et al.
(författare)
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Inhibition of nSMase2 Reduces the Transfer of Oligomeric alpha-Synuclein Irrespective of Hypoxia
- 2019
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Ingår i: Frontiers in Molecular Neuroscience. - : FRONTIERS MEDIA SA. - 1662-5099. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Recently, extracellular vesicles (EVs), such as exosomes, have been proposed to play an influential role in the cell-to-cell spread of neurodegenerative diseases, including the intercellular transmission of alpha-synuclein (alpha-syn). However, the regulation of EV biogenesis and its relation to Parkinson's disease (PD) is only partially understood. The generation of EVs through the ESCRT-independent pathway depends on the hydrolysis of sphingomyelin by neutral sphingomyelinase 2 (nSMase2) to produce ceramide, which causes the membrane of endosomal multivesicular bodies to bud inward. nSMase2 is sensitive to oxidative stress, a common process in PD brains; however, little is known about the role of sphingomyelin metabolism in the pathogenesis of PD. This is the first study to show that inhibiting nSMase2 decreases the transfer of oligomeric aggregates of alpha-syn between neuron-like cells. Furthermore, it reduced the accumulation and aggregation of high-molecular-weight alpha-syn. Hypoxia, as a model of oxidative stress, reduced the levels of nSMase2, but not its enzymatic activity, and significantly altered the lipid composition of cells without affecting EV abundance or the transfer of alpha-syn. These data show that altering sphingolipids can mitigate the spread of alpha-syn, even under hypoxic conditions, potentially suppressing PD progression.
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| 4. |
- Sackmann, Valerie, 1990-
(författare)
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The Propagation of Neurodegenerative Diseases by Inflammation and Exosomes
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases with rates increasing along with the ageing global population. Despite best efforts, we still do not understand the etiopathogenesis of these diseases and there are no effective disease-modifying treatments. Cognitive deficiencies or motor complications that emerge during AD and PD are thought to be the result of the accumulation of misfolded, aggregate-prone proteins, such as amyloid-β (Aβ) and tau or α-synuclein (α-syn), respectively. Growing evidence suggests that prefibrillar oligomers of Aβ and α-syn (oAβ and oα-syn) are key contributors to the progression of these diseases. The progressive accumulation of these proteins leads to a gradual spread of pathology throughout interconnected brain regions, but the mechanisms by which this spreading occurs are still largely unknown.Neuroinflammation has been recognised as an important contributor to neurodegenerative disease. It is hypothesised that a pro-inflammatory environment initiated by the innate immune system, either through activation from Aβ itself or indirectly through neuronal injury signals in AD. These phenomena are thought to either cause or accelerate AD, such that an anti-inflammatory approach may be neuroprotective. In paper I, we investigated whether different inflammatory environments affected the transfer of oAβ between neuron-like cells, in addition to investigating inter- and intracellular protein changes. This study demonstrated that an anti-inflammatory environment reduces the transfer of oAβ between cells. We also provide evidence that these cells begin to take on the “phenotype” of the inflammatory milieu, while also demonstrating that the expression profile of endosomal/lysosomal and protein trafficking proteins is altered during these conditions.Small extracellular vesicles called exosomes, which are key players in cell to cell communication, have been proposed to play an influential role in spreading neurodegenerative proteins between cells. Exosomes are small membranous vesicles that are formed by the inward budding of multivesicular bodies (MVBs). These MVBs can then merge with the plasma membrane to be released into the extracellular environment as vesicles, which serve as vehicles for transferring proteins, lipids, and mRNAs between cells.The ESCRT-dependent pathway is the most understood mechanism underlying exosome biogenesis. However, exosomes can also be formed through ESCRT-independent pathways, including through the hydrolysis of sphingomyelin by neutral sphingomyelinase 2 (nSMase2), which produces ceramide. Paper II investigated whether exosomes formed through an ESCRT-independent pathway plays a significant role in the transfer of oα-syn between neuron-like cells. As oxidative stress is a common feature in PD brains, which in turn dysregulates nSMase2 activity, we also tested our model under hypoxic conditions. Inhibition of nSMase2 significantly reduced the transfer of oα-syn between cells but also resulted in decreased α-syn aggregation. Hypoxia did not influence oα-syn transfer, however, it significantly dysregulated the sphingolipid composition, which may be important for α-syn binding to exosomes and exosome communication.During AD and PD, there is a noted reduction in the effectiveness of autophagy, a process critical to cellular proteostasis. Recent studies have uncovered shared regulatory mechanisms of exosome biogenesis and autophagy, suggesting that they are closely linked. Previous findings have shown that inhibition of autophagy in AD mice mediates Aβ trafficking through altering the secretion of Aβ in MVBs. To further study this effect, we investigated the interplay between autophagy and exosome secretion using ATG7 knock-out x APPNL-F knock-in AD mice in paper III. These autophagy-deficient AD mice had a reduced extracellular Aβ plaque load, but increased intracellular Aβ, which was found to be assembled into higher-ordered assemblies. While exosomal secretion was dysregulated in these mice, the amount of Aβ packaged into the exosomes was unchanged.Lastly, one of the biggest challenges in developing effective treatments for AD is the lack of early diagnosis of living patients. As the connection between exosomes and the spread of neurodegenerative proteins is still relatively new, there remains a diagnostic potential to be explored with exosomes. Paper IV aimed to develop a new diagnostic assay to detect oAβ in exosomes isolated from human cerebrospinal fluid. Although exosomal oAβ was readily detected in some of these samples, the assay’s sensitivity requires additional optimisation before it can be further validated for the clinic.In summary, the studies presented in this thesis have furthered our understanding of how inflammation, autophagy, and exosomes contribute to the intercellular transmission of AD and PD associated proteins. We have shown that an anti-inflammatory approach may slow down the progression of AD through reducing the transfer of oAβ between cells. We also provide novel findings relating to the biogenesis of exosomes, which in turn affected the ability of exosomes to transmit neurodegenerative proteins between cells, and their association with autophagic processes. Finally, we have investigated the feasibility of exosomes as an early AD diagnostic marker. This work has helped to elucidate some of the mechanisms underlying the progression of neurodegenerative diseases, which may be useful targets for the investigation of new therapeutic avenues.
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| 5. |
- Sardar Sinha, Maitrayee, et al.
(författare)
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Alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers
- 2018
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Ingår i: Acta Neuropathologica. - : SPRINGER. - 0001-6322 .- 1432-0533. ; 136:1, s. 41-56
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Tidskriftsartikel (refereegranskat)abstract
- The gradual deterioration of cognitive functions in Alzheimer's disease is paralleled by a hierarchical progression of amyloid-beta and tau brain pathology. Recent findings indicate that toxic oligomers of amyloid-beta may cause propagation of pathology in a prion-like manner, although the underlying mechanisms are incompletely understood. Here we show that small extracellular vesicles, exosomes, from Alzheimer patients' brains contain increased levels of amyloid-beta oligomers and can act as vehicles for the neuron-to-neuron transfer of such toxic species in recipient neurons in culture. Moreover, blocking the formation, secretion or uptake of exosomes was found to reduce both the spread of oligomers and the related toxicity. Taken together, our results imply that exosomes are centrally involved in Alzheimer's disease and that they could serve as targets for development of new diagnostic and therapeutic principles.
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| 6. |
- Wiechec, Emilia, et al.
(författare)
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Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma
- 2021
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:13
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Cancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and have been shown to stimulate tumor growth, epithelial-to-mesenchymal transition (EMT), invasion, and radio-resistance. Radio-resistance leading to disease relapse is one of the major challenges in long-term survival and outcome in head and neck squamous cell carcinoma (HNSCC). Therefore, it is essential to search for predictive markers and new targets for treatment using clinically relevant in vitro tumor models. We show that CAFs alter the expression of HNSCC tumor cell genes, many of which are associated with proliferation, differentiation, and metastasis. Moreover, the expression pattern of selected CAF-regulated genes differed between HNSCC tumor tissue and the adjacent non-tumoral tissue. Two CAF-regulated genes, MMP9 and FMOD, were found to be associated with overall survival (OS) in patients treated with radiotherapy. Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan-Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.
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