| 1. |
- Beretta, Chiara, et al.
(författare)
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Amyloid-β deposits in human astrocytes contain truncated and highly resistant proteoforms
- 2024
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 128
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) is a neurodegenerative disorder that develops over decades. Glial cells, including astrocytes are tightly connected to the AD pathogenesis, but their impact on disease progression is still unclear. Our previous data show that astrocytes take up large amounts of aggregated amyloid-beta (Aβ) but are unable to successfully degrade the material, which is instead stored intracellularly. The aim of the present study was to analyze the astrocytic Aβ deposits composition in detail in order to understand their role in AD propagation. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and magnetic beads. Live cell imaging and immunocytochemistry confirmed that the ingested Aβ aggregates and beads were transported to the same lysosomal compartments in the perinuclear region, which allowed us to successfully isolate the Aβ deposits from the astrocytes. Using a battery of experimental techniques, including mass spectrometry, western blot, ELISA and electron microscopy we demonstrate that human astrocytes truncate and pack the Aβ aggregates in a way that makes them highly resistant. Moreover, the astrocytes release specifically truncated forms of Aβ via different routes and thereby expose neighboring cells to pathogenic proteins. Taken together, our study establishes a role for astrocytes in mediating Aβ pathology, which could be of relevance for identifying novel treatment targets for AD.
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| 2. |
- Beretta, Chiara, 1992-
(författare)
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Astrocytes in Alzheimer’s disease : Exploring the impact of amyloid-β pathology on neurotoxicity, metabolism and inflammation.
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Astrocytes play a central role in brain homeostasis, but are also tightly connected to the pathogenesis of Alzheimer’s disease (AD). Yet, their exact role in amyloid-beta (Aβ) pathology and chronic neuroinflammation is unclear. The aim of this thesis was to elucidate the impact of astrocytes in AD progression. For this purpose, astrocytes in different culture set-ups were exposed to soluble Aβ aggregates. The astrocytes engulf and process, but fail to fully degrade the Aβ aggregates, which are instead stored as large intracellular deposits. In Paper I, we show that extracellular vesicles (EVs), secreted from the Aβ-containing cells induce synaptic loss, axonal swelling and vacuolization of primary neurons, which consequently leads to apoptosis. Astrocytes play a central role in the brain’s energy metabolism and we were therefore interested in how Aβ pathology affects their metabolism. In Paper II, we report that Aβ accumulation in astrocytes disrupts mitochondrial fission/fusion homeostasis, resulting in decreased mitochondrial respiration and altered glycolysis. Interestingly, the astrocytes switch to fatty acid β oxidation with the aid of peroxisomes to maintain stable energy production. Another important task is to understand how astrocytes modify the ingested Aβ. In Paper III, we characterized the astrocytic Aβ inclusions by isolating them with magnetic beads. Our analysis showed that the astrocytes truncate and pack together the Aβ aggregates. Moreover, we found that astrocytes release specifically truncated forms of Aβ via different routes.Astrocytes’ involvement in lipid metabolism and inflammation has recently gained much interest, but many questions remain about the connection between these processes. In Paper IV, we show that Aβ pathology causes lipid droplet (LD) accumulation in astrocytes. Moreover, we could show that astrocytes frequently transfer LDs to neighboring cells, both through direct cell-to-cell contacts and via secretion. Astrocytes have previously been reported to express major histocompatibility complex II (MHCII) and have the capacity to perform as professional antigen presenting cells. Interestingly, our results demonstrate that LDs contain MHCII, identifying a link between LDs and inflammation in astrocytes.Taken together, this thesis contributes with important knowledge of the role of astrocytes in AD pathology.
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| 3. |
- Beretta, Chiara, et al.
(författare)
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Astrocytes with Alzheimer’s disease pathology provoke lipid droplet mediated cell-to-cell propagation of MHC II complexes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background. Astrocytes are critical for maintaining brain homeostasis, but are also highly involved in neuroinflammation. In the Alzheimer disease (AD) brain, reactive, inflammatory astrocytes are situated closely around amyloid β (Aβ) plaques. We have previously shown that reactive astrocytes ingest large quantities of soluble Aβ aggregates, but are unable to degrade the material, which leads to intracellular Aβ accumulation and severe cellular stress. A common response to cellular stress is the formation of lipid droplets (LDs). Novel data indicate that LDs play an important role in inflammatory processes. However, the involvement of LDs in AD inflammation and progression remains unclear.Methods. The aim of this study was to investigate how astrocytic Aβ pathology affects lipid metabolism and antigen presentation. For this purpose, human induced pluripotent stem cell (iPSC) derived astrocytes were exposed to soluble Aβ42 aggregates and analyzed over time, using a battery of experimental approaches.Results. Our results show that Aβ exposure induces LD accumulation in astrocytes, although the overall lipid composition remains unchanged. Moreover, astrocytes transfer LDs to neighboring cells via tunneling nanotubes (TNTs) and extracellular vesicle (EVs). Interestingly, we found that the antigen presenting protein major histocompatibility complex II (MHCII) is present inside LDs, suggesting an active role of LDs in astrocytic antigen presentation. Immunohistochemical analysis of human brain tissue verified the presence of LD-loaded MHCII+ astrocytes in AD individuals. Moreover, we found infiltrated CD4+ T cells to be in close contact with astrocytes, confirming an astrocyte T cell cross-talk in the AD brainConclusions. Taken together, our data show that Aβ pathology drastically affects lipid storage in astrocytes, which in turn modulates the astrocytic antigen presentation, indicating a role for astrocytic LDs in T cell responses in the AD brain.
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| 4. |
- Beretta, Chiara, et al.
(författare)
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Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons
- 2020
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Ingår i: Scientific Reports. - : NATURE RESEARCH. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-beta (A beta) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of A beta (42) protofibrils by astrocytes results in the release of EVs containing neurotoxic A beta. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and A beta (42) protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from A beta (42) protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from A beta exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of A beta -induced pathology.
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| 5. |
- Konstantinidis, Evangelos, 1990-, et al.
(författare)
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Amyloid-beta accumulation in astrocytes affects their impact on neuronal function in a human iPSC-based model of Alzheimer’s disease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Although Alzheimer’s disease (AD) is the leading cause of dementia worldwide, there are currently no treatments available that limit the neurodegeneration or slow down the disease progression. Hence, innovative therapeutic approaches are clearly required. The role of astrocytes in AD has recently received much attention, due to their central function in brain homeostasis and synaptic function. Accumulating evidence indicates that astrocytes may lose the ability to fulfil some of their physiological tasks when shifting towards an inflammatory state. Our previous data demonstrate that astrocytes can ingest large amounts of aggregated amyloid-beta (Aβ), but then store, rather than degrade, the ingested material. This incomplete degradation results in severe cellular stress, which could be of relevance for AD progression.Methods: In this study, we aimed to investigate how inclusions of aggregated Aβ in astrocytes affect their interplay with neurons, focusing on cellular viability and synaptic function. For this purpose, human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Αβ42 fibrils and their impact on hiPSC-derived neurons was analyzed by performing neuron-astrocyte co-cultures as well as additions of conditioned media or extracellular vesicles to pure neuronal cultures.Results: In the co-culture setup, the presence of Aβ inclusions led to an elevated clearance of dead cells by the astrocytes, indicating increased glial reactivity. In contrast, conditioned media from control, but not from Aβ-exposed astrocytes, benefited the wellbeing of neuronal monocultures. Furthermore, electrophysiological recordings showed a significant decrease in the frequency of excitatory post synaptic current (sEPSCs) in neurons co-cultured with Aβ-astrocytes compared to control astrocytes, while conditioned media from Aβ-exposed astrocytes had the opposite effect.Conclusions: Taken together, our results demonstrate that inclusions of aggregated Aβ affect the reactivity state of astrocytes, as well as their ability to support neuronal function.
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| 6. |
- Konstantinidis, Evangelos, 1990-, et al.
(författare)
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Intracellular deposits of amyloid-beta influence the ability of human iPSC-derived astrocytes to support neuronal function
- 2023
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Ingår i: Journal of Neuroinflammation. - : Springer Nature. - 1742-2094. ; 20:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundAstrocytes are crucial for maintaining brain homeostasis and synaptic function, but are also tightly connected to the pathogenesis of Alzheimer’s disease (AD). Our previous data demonstrate that astrocytes ingest large amounts of aggregated amyloid-beta (Aβ), but then store, rather than degrade the ingested material, which leads to severe cellular stress. However, the involvement of pathological astrocytes in AD-related synaptic dysfunction remains to be elucidated.MethodsIn this study, we aimed to investigate how intracellular deposits of Aβ in astrocytes affect their interplay with neurons, focusing on neuronal function and viability. For this purpose, human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Αβ42 fibrils. The direct and indirect effects of the Αβ-exposed astrocytes on hiPSC-derived neurons were analyzed by performing astrocyte–neuron co-cultures as well as additions of conditioned media or extracellular vesicles to pure neuronal cultures.ResultsElectrophysiological recordings revealed significantly decreased frequency of excitatory post-synaptic currents in neurons co-cultured with Aβ-exposed astrocytes, while conditioned media from Aβ-exposed astrocytes had the opposite effect and resulted in hyperactivation of the synapses. Clearly, factors secreted from control, but not from Aβ-exposed astrocytes, benefited the wellbeing of neuronal cultures. Moreover, reactive astrocytes with Aβ deposits led to an elevated clearance of dead cells in the co-cultures.ConclusionsTaken together, our results demonstrate that inclusions of aggregated Aβ affect the reactive state of the astrocytes, as well as their ability to support neuronal function.
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| 7. |
- Konstantinidis, Evangelos, 1990-, et al.
(författare)
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Long-term effects of amyloid-beta accumulation in human iPSC-derived astrocytes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Alzheimer's disease (AD) is a slowly progressive condition and the most common cause of dementia worldwide. The pathological changes associated with the disease are estimated to occur decades before the first clinical symptoms emerge. Although neurons have been the main research focus, astrocytes have recently gained attention because of their participation in several key cellular functions. Our previous data suggest that astrocytes engulf large amounts of aggregated amyloid-beta (Aβ), but are unable to successfully degrade the material. The intracellular accumulation is, at least initially, very stressful for the astrocytes and long-term Aβ deposits may be detrimental for their functionality. However, if the astrocytes can handle the Aβ storage it could instead be beneficial in a longer perspective, by minimizing the spread of potentially pathogenic Aβ species to neighboring cells.Methods: We aimed to evaluate the effects of long-term Aβ inclusions in astrocytes. Mature human induced pluripotent stem cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and then further cultured for one week or ten weeks in Aβ-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the medium was screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy.Results: Long-term astrocytes displayed frequent Aβ inclusions that were contained within LAMP1 positive organelles and sustained markers associated with reactivity. Furthermore, increased levels of CCL2/MCP-1 were detected in the media from Aβ-exposed long-term cultures. Finally, Aβ accumulation induced endoplasmic reticulum and mitochondrial swelling as well as formation of pathological lipid structures in astrocytes.Conclusions: This study provides valuable information about how intracellular Aβ deposits affect astrocytes over time, and thereby contribute to the understanding of astrocytes role in AD progression.
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| 8. |
- Konstantinidis, Evangelos, 1990-, et al.
(författare)
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Long-term effects of amyloid-beta deposits in human iPSC-derived astrocytes
- 2023
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 125
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Tidskriftsartikel (refereegranskat)abstract
- Growing evidence indicates that astrocytes are tightly connected to Alzheimer's disease (AD) pathogenesis. However, the way in which astrocytes participate in AD initiation and progression remains to be clarified. Our previous data show that astrocytes engulf large amounts of aggregated amyloid-beta (A beta) but are unable to successfully degrade the material. In this study, we aimed to evaluate how intracellular A beta-accumulation affects the astrocytes over time. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated A beta-fibrils and then cultured further for one week or ten weeks in A beta-free medium. Cells from both time points were analyzed for lysosomal proteins and astrocyte reactivity markers and the media were screened for inflammatory cytokines. In addition, the overall health of cytoplasmic organelles was investigated by immunocytochemistry and electron microscopy. Our data demonstrate that long-term astrocytes retained frequent A beta-inclusions that were enclosed within LAMP1-positive organelles and sustained markers associated with reactivity. Furthermore, A beta-accumulation resulted in endoplasmic reticulum and mitochondrial swelling, increased secretion of the cytokine CCL2/MCP-1 and formation of pathological lipid structures. Taken together, our results provide valuable information of how intracellular A beta-deposits affect astrocytes, and thereby contribute to the understanding of the role of astrocytes in AD progression.
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| 9. |
- Lepri, Gemma, et al.
(författare)
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Systemic sclerosis and primary biliary cholangitis : Longitudinal data to determine the outcomes
- 2023
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Ingår i: Journal of Scleroderma and Related Disorders. - 2397-1983. ; 8:3, s. 210-220
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Tidskriftsartikel (refereegranskat)abstract
- Background: Several studies described the cross-sectional characteristics of systemic sclerosis patients and coexisting primary biliary cholangitis, but longitudinal prognostic data are lacking. Aims: To describe the systemic sclerosis–primary biliary cholangitis phenotype, including baseline characteristics and outcomes. Methods: We performed a multicentre the European Scleroderma Trials and Research Group study of systemic sclerosis patients with primary biliary cholangitis or with primary biliary cholangitis–specific antibodies, matched with systemic sclerosis controls free from hepatobiliary involvement matched for disease duration and cutaneous subset. Data were recorded at baseline and at the last available visit. Results: A total of 261 patients were enrolled (115 primary biliary cholangitis–systemic sclerosis, 161 systemic sclerosis). At baseline, systemic sclerosis–primary biliary cholangitis patients had a higher prevalence of anti-centromere antibodies (p = 0.0023) and a lower prevalence of complete absence of digital ulcers. The milder vascular involvement was confirmed at follow-up when crucial differences emerged in the percentage of patients experiencing digital ulcers; a significantly higher number of patients who never experienced digital ulcers were observed among primary biliary cholangitis–systemic sclerosis patients (p = 0.0015). Moreover, a greater incidence of pulmonary arterial hypertension (p < 0.001) and of conduction blocks (p = 0.0256) was observed in systemic sclerosis patients without primary biliary cholangitis. Patients with primary biliary cholangitis had higher levels of liver enzymes at baseline than systemic sclerosis patients; a significant decrease in liver enzymes was observed at follow-up. Out of 18 patients with cholangitis, one received a liver transplant at follow-up. Conclusion: Our data show that systemic sclerosis–primary biliary cholangitis exhibit a mild systemic sclerosis and primary biliary cholangitis phenotype with outcomes being in general favourable.
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| 10. |
- López-Isac, Elena, et al.
(författare)
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Brief Report : IRF4 Newly Identified as a Common Susceptibility Locus for Systemic Sclerosis and Rheumatoid Arthritis in a Cross-Disease Meta-Analysis of Genome-Wide Association Studies
- 2016
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Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 68:9, s. 2338-2344
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Systemic sclerosis (SSc) and rheumatoid arthritis (RA) are autoimmune diseases that have similar clinical and immunologic characteristics. To date, several shared SSc–RA genetic loci have been identified independently. The aim of the current study was to systematically search for new common SSc–RA loci through an interdisease meta–genome-wide association (meta-GWAS) strategy. Methods: The study was designed as a meta-analysis combining GWAS data sets of patients with SSc and patients with RA, using a strategy that allowed identification of loci with both same-direction and opposite-direction allelic effects. The top single-nucleotide polymorphisms were followed up in independent SSc and RA case–control cohorts. This allowed an increase in the sample size to a total of 8,830 patients with SSc, 16,870 patients with RA, and 43,393 healthy controls. Results: This cross-disease meta-analysis of the GWAS data sets identified several loci with nominal association signals (P < 5 × 10−6) that also showed evidence of association in the disease-specific GWAS scans. These loci included several genomic regions not previously reported as shared loci, as well as several risk factors that were previously found to be associated with both diseases. Follow-up analyses of the putatively new SSc–RA loci identified IRF4 as a shared risk factor for these 2 diseases (Pcombined = 3.29 × 10−12). Analysis of the biologic relevance of the known SSc–RA shared loci identified the type I interferon and interleukin-12 signaling pathways as the main common etiologic factors. Conclusion: This study identified a novel shared locus, IRF4, for the risk of SSc and RA, and highlighted the usefulness of a cross-disease GWAS meta-analysis strategy in the identification of common risk loci.
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