| 1. |
- Domert, Jakob, et al.
(författare)
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Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes
- 2016
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein.
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| 2. |
- Gustafsson, Gabriel, et al.
(författare)
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Secretion and uptake of α-synuclein via extracellular vesicles in cultured cells
- 2018
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Ingår i: Cellular and molecular neurobiology. - : Springer Science and Business Media LLC. - 0272-4340 .- 1573-6830. ; 38:8, s. 1539-1550
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Tidskriftsartikel (refereegranskat)abstract
- In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.
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| 3. |
- Mosrati, Mohamed Ali, et al.
(författare)
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TERT promoter mutations and polymorphisms as prognostic factors in primary glioblastoma
- 2015
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Ingår i: Oncotarget. - : IMPACT JOURNALS LLC. - 1949-2553. ; 6:18, s. 16663-16673
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Tidskriftsartikel (refereegranskat)abstract
- Telomerase reverse transcriptase (TERT) activity is up-regulated in several types of tumors including glioblastoma (GBM). In the present study, 128 primary glioblastoma patients were examined for single nucleotide polymorphisms of TERT in blood and in 92 cases for TERT promoter mutations in tumors. TERT promoter mutations were observed in 86% of the tumors and of these, C228T (-124 bp upstream start codon) was detected in 75% and C250T (-146 bp) in 25% of cases. TERT promoter mutations were associated with shorter overall survival (11 vs. 20 months p = 0.002 and 12 vs. 20, p = 0.04 for C228T and C250T, respectively). The minor alleles of rs2736100 and rs10069690 SNPs, located in intron 2 and the promotor regions, respectively, were associated with an increased risk of developing GBM (p = 0.004 and 0.001). GBM patients having both TERT promoter mutations and being homozygous carriers of the rs2853669 C-allele displayed significantly shorter overall survival than those with the wild type allele. The rs2853669 SNP is located in a putative Ets2 binding site in the promoter (-246 bp upstream start codon) close to the C228T and C250T mutation hot spots. Interleukin-6 (IL-6) expression regulated by TERT promoter status and polymorphism, what leads us to think that TERT and IL-6 plays a significant role in GBM, where specific SNPs increase the risk of developing GBM while the rs2853669 SNP and specific mutations in the TERT promoter of the tumor lead to shorter survival.
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| 4. |
- Reyes, Juan, et al.
(författare)
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Binding of alfa-synuclein oligomers to Cx32 facilitates protein uptake and transfer in neurons and oligodendrocytes
- 2019
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Ingår i: Acta Neuropathologica. - : SPRINGER. - 0001-6322 .- 1432-0533. ; 138:1, s. 23-47
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Tidskriftsartikel (refereegranskat)abstract
- The intercellular transfer of alpha-synuclein (-syn) has been implicated in the progression of Parkinson's disease (PD) and multiple system atrophy (MSA). The cellular mechanisms underlying this process are now beginning to be elucidated. In this study, we demonstrate that the gap junction protein connexin-32 (Cx32) is centrally involved in the preferential uptake of -syn oligomeric assemblies (o-syn) in neurons and oligodendrocytes. In vitro, we demonstrate a clear correlation between Cx32 expression and o-syn uptake. Pharmacological and genetic strategies targeting Cx32 successfully blocked o-syn uptake. In cellular and transgenic mice modeling PD and MSA, we observed significant upregulation of Cx32 which correlates with -syn accumulation. Notably, we could alsodemonstrate a direct interaction between -syn and Cx32 in two out of four human PD cases that was absent in all four age-matched controls. These data are suggestive of a link between Cx32 and PD pathophysiology. Collectively, our results provide compelling evidence for Cx32 as a novel target for therapeutic intervention in PD and related -synucleinopathies.
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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. |
- 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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| 7. |
- Bruhn, H., et al.
(författare)
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Improved survival of Swedish glioblastoma patients treated according to Stupp
- 2018
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Ingår i: Acta Neurologica Scandinavica. - : WILEY. - 0001-6314 .- 1600-0404. ; 138:4, s. 332-337
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesThe median survival in glioblastoma (GBM) patients used to be less than 1year. Surgical removal of the tumor with subsequent concomitant radiation/temozolomide (the Stupp regimen) has been shown to prolong survival. The Stupp protocol was implemented in the county of Jonkoping in 2006. The purpose of this study was to examine if the Stupp treatment has prolonged overall survival, in an unselected patient cohort with histologically verified GBM. Material and MethodThis study includes all patients from the county of Jonkoping, with a diagnosis of GBM from January 2001 to December 2012. Patients were divided into 2 cohorts, 2001-2005 and 2006-2012, that is before and after implementation of the Stupp regimen. By reviewing the medical case notes, the dates of the histological diagnosis and of death were identified. The median and mean overall survival and Kaplan-Meier survival analysis were calculated and compared between the 2 cohorts. ResultsThe mean survival was 110days longer in the cohort treated according to the Stupp regimen. Four patients in the 2006-2012 cohort and 1 patient in the 2001-2005 cohort are still alive. When comparing survival in patients with radical surgery vs biopsy, those that underwent radical surgery survived longer. The significance was slightly greater in the 2001-2005 cohort (mean 163 vs 344days, Pamp;lt;.001) than in the 2006-2012 cohort (mean 220 vs 397days, P=.02). ConclusionSurvival significantly improved after the implementation of the Stupp regimen in the study region of Sweden.
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| 8. |
- Domert, Jakob, 1986-
(författare)
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Neuron-to-neuron propagation of neurodegenerative proteins; relation to degradative systems
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are defined by neurodegeneration and accumulations of misfolded proteins that spread through the brain in a well characterized manner. In AD these accumulations consist mainly of β-amyloid (Aβ) and tau, while in PD, α-synuclein (α-syn) make up the characteristic lewy pathology. The general aim of this thesis was to investigate mechanisms associated with neurotoxic peptide activity by Aβ, tau and α-syn in relation to cellular degradation and transfer with a cell-to-cell transfer model system. We found that intercellular transfer of oligomeric Aβ occurs independently of isoform. However, the amount of transfer correlates with each isoforms ability to resist degradation or cellular clearance. The Aβ1-42 isoform showed particular resistance to clearance, which resulted in higher levels of cell-to-cell transfer of the isoform and lysosomal stress caused by accumulation. As Aβ accumulations can inhibit the proteasomal degradation we investigated how reduced proteasomal degradation affected neuron-like cells. We found increased levels of phosphorylated tau protein, disturbed microtubule stability and impaired neuritic transport after reduced proteasomal activity. These changes was partly linked to c-Jun and ERK 1/2 kinase activity. We could also show that α-syn transferred from cell-to-cell in our model system, with a higher degree of transfer for the larger oligomer and fibrillar species. Similar to Aβ, α-syn mainly colocalized with lysosomes, before and after transfer. Lastly, we have developed our cell-to-cell transfer system into a model suitable for high throughput screening (HTS). The type of cells have been upgraded from SH-SY5Y cells to induced pluripotent stem cells (iPSCs), with a differentiation profile more similar to mature neurons. The next step will be screening a small molecular library for substances with inhibitory effect on cell-to-cell transfer of Aβ peptides. The importance of the degradative systems in maintaining protein homeostasis and prevent toxic accumulations in general is well known. Our findings shows the importance of these systems for neurodegenerative diseases and also highlight the link between degradation and cell-to-cell transfer. To restore or enhance the degradative systems would be an interesting avenue to treat neurodegenerative diseases. Another way would be to inhibit the transfer of misfolded protein aggregates. By using the HTS model we developed, a candidate substance with good inhibitory effect on transfer can hopefully be found.
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| 9. |
- Gustafsson, Håkan, et al.
(författare)
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Visualization of oxidative stress in ex vivo biopsies using electron paramagnetic resonance imaging
- 2015
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Ingår i: Magnetic Resonance in Medicine. - : Wiley. - 0740-3194 .- 1522-2594. ; 73:4, s. 1682-1691
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: The purpose of this study was to develop an X-Band electron paramagnetic resonance imaging protocol for visualization of oxidative stress in biopsies.METHODS: The developed electron paramagnetic resonance imaging protocol was based on spin trapping with the cyclic hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine and X-Band EPR imaging. Computer software was developed for deconvolution and back-projection of the EPR image. A phantom containing radicals of known spatial characteristic was used for evaluation of the developed protocol. As a demonstration of the technique electron paramagnetic resonance imaging of oxidative stress was performed in six sections of atherosclerotic plaques. Histopathological analyses were performed on adjoining sections.RESULTS: The developed computer software for deconvolution and back-projection of the EPR images could accurately reproduce the shape of a phantom of known spatial distribution of radicals. The developed protocol could successfully be used to image oxidative stress in six sections of the three ex vivo atherosclerotic plaques.CONCLUSIONS: We have shown that oxidative stress can be imaged using a combination of spin trapping with the cyclic hydroxylamine spin probe cyclic hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine and X-Band EPR imaging. A thorough and systematic evaluation on different types of biopsies must be performed in the future to validate the proposed technique. Magn Reson Med, 2014.
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| 10. |
- Haj-Hosseini, Neda, 1980-, et al.
(författare)
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5-ALA fluorescence and laser Doppler flowmetry for guidance in a stereotactic brain tumor biopsy
- 2018
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Ingår i: Biomedical Optics Express. - : Optical Society of America. - 2156-7085. ; 9:5, s. 2284-2296
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Tidskriftsartikel (refereegranskat)abstract
- A fiber optic probe was developed for guidance during stereotactic brain biopsy procedures to target tumor tissue and reduce the risk of hemorrhage. The probe was connected to a setup for the measurement of 5-aminolevulinic acid (5-ALA) induced fluorescence and microvascular blood flow. Along three stereotactic trajectories, fluorescence (n = 109) and laser Doppler flowmetry (LDF) (n = 144) measurements were done in millimeter increments. The recorded signals were compared to histopathology and radiology images. The median ratio of protoporphyrin IX (PpIX) fluorescence and autofluorescence (AF) in the tumor was considerably higher than the marginal zone (17.3 vs 0.9). The blood flow showed two high spots (3%) in total. The proposed setup allows simultaneous and real-time detection of tumor tissue and microvascular blood flow for tracking the vessels.
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