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Cellular responses to amyloid-beta protofibrils: Focus on astrocytes, extracellular vesicles and antibody treatment
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Knowledge about the cellular mechanisms behind the initiation and propagation of Alzheimer’s disease (AD) is limited. Decades of research have focused on neuronal abnormalities in AD, but recently more attention has been given to the glial cells. Being the most numerous glial cell type in the brain, astrocytes are important for many functions, but their role in AD is poorly understood. The aim with this thesis was to clarify the involvement of astrocytes in AD by using a co-culture system of primary neurons and glia. The co-cultures were exposed to soluble amyloid-beta (Aβ) aggregates, i.e. protofibrils that are known to be particularly harmful.

In Paper I, the capacity of astrocytes to ingest and degrade Aβ protofibrils was investigated. We found that astrocytes effectively ingested Aβ, but were ineffective in degrading the material. The intracellular accumulation of Aβ in astrocytes resulted in lysosomal dysfunction, high intracellular load of partly N-terminally truncated Aβ and extracellular vesicle (EV) mediated neuronal cell death.

Cells can communicate by releasing cargo into EVs, but the role of EVs in the spreading of Aβ pathology is unclear. In Paper II, the protein content of EVs released specifically following Aβ protofibril exposure was analyzed. We found markedly increased levels of apolipoprotein E (apoE) in EVs from Aβ protofibril exposed co-cultures, suggesting a role for intercellular transfer of apoE in Aβ pathology.

Passive immunotherapy has been suggested as a promising therapeutic strategy for AD. In Paper III, we investigated if the Aβ protofibril-selective antibody mAb158 could affect Aβ clearance in the co-culture. The mAb158 treatment reduced Aβ accumulation in astrocytes and rescued neurons from Aβ-induced cell death.

In Paper IV, we explored the effect of EVs, isolated from Aβ protofibril exposed co-cultures on cultured neurons. In addition to increased cell death, we found that such EVs had a strong negative impact on the synapses, dendrites and mitochondria of the neurons.

Taken together, this thesis contributes with important knowledge about the role of astrocytes in Aβ pathology, the vesicle-mediated spreading of Aβ and the effects of anti-Aβ antibody treatment.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1405
Keywords [en]
Alzheimer’s disease, Amyloid-beta, Protofibrils, Astrocytes, Extracellular vesicles, Antibody, Neurons, Degradation
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-334358ISBN: 978-91-513-0173-0 (print)OAI: oai:DiVA.org:uu-334358DiVA, id: diva2:1161000
Public defence
2018-02-09, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-01-17 Created: 2017-11-28 Last updated: 2018-03-07
List of papers
1. Accumulation of amyloid-beta by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons
Open this publication in new window or tab >>Accumulation of amyloid-beta by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons
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2016 (English)In: Molecular Neurodegeneration, ISSN 1750-1326, E-ISSN 1750-1326, Vol. 11, article id 38Article in journal (Refereed) Published
Abstract [en]

Background: Despite the clear physical association between activated astrocytes and amyloid-beta (A beta) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer's disease remain elusive. Soluble A beta aggregates, such as protofibrils, have been suggested to be responsible for the widespread neuronal cell death in Alzheimer's disease, but the mechanisms behind this remain unclear. Moreover, ineffective degradation is of great interest when it comes to the development and progression of neurodegeneration. Based on our previous results that astrocytes are extremely slow in degrading phagocytosed material, we hypothesized that astrocytes may be an important player in these processes. Hence, the aim of this study was to clarify the role of astrocytes in clearance, spreading and neuronal toxicity of A beta. Results: To examine the role of astrocytes in A beta pathology, we added A beta protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of A beta protofibrils, but then store, rather than degrade the ingested material. The incomplete digestion results in a high intracellular load of toxic, partly N-terminally truncated A beta and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated A beta, induce apoptosis of cortical neurons. Conclusions: Taken together, our results suggest that astrocytes play a central role in the progression of Alzheimer's disease, by accumulating and spreading toxic A beta species.

Keywords
Alzheimer's disease, Protofibrils, Glia, Phagocytosis, Degradation, Enlarged vacuole, Microvesicle
National Category
Neurology
Identifiers
urn:nbn:se:uu:diva-298408 (URN)10.1186/s13024-016-0098-z (DOI)000376366500001 ()27176225 (PubMedID)
Available from: 2016-07-04 Created: 2016-07-04 Last updated: 2017-11-28Bibliographically approved
2. Increased Release of Apolipoprotein E in Extracellular Vesicles Following Amyloid-β Protofibril Exposure of Neuroglial Co-Cultures
Open this publication in new window or tab >>Increased Release of Apolipoprotein E in Extracellular Vesicles Following Amyloid-β Protofibril Exposure of Neuroglial Co-Cultures
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2017 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 60, no 1, p. 305-321Article in journal (Refereed) Published
Abstract [en]

Extracellular vesicles (EVs), including exosomes and larger microvesicles, have been implicated to play a role in several conditions, including Alzheimer's disease (AD). Since the EV content mirrors the intracellular environment, it could contribute with important information about ongoing pathological processes and may be a useful source for biomarkers, reflecting the disease progression. The aim of the present study was to analyze the protein content of EVs specifically released from a mixed co-culture of primary astrocytes, neurons, and oligodendrocytes treated with synthetic amyloid-beta (A beta(42)) protofibrils. The EV isolation was performed by ultracentrifugation and validated by transmission electron microscopy. Mass spectrometry analysis of the EV content revealed a total of 807 unique proteins, of which five displayed altered levels in A beta(42) protofibril exposed cultures. The most prominent protein was apolipoprotein E (apoE), and by western blot analysis we could confirm a threefold increase of apoE in EVs from A beta(42) protofibril exposed cells, compared to unexposed cells. Moreover, immunoprecipitation studies demonstrated that apoE was primarily situated inside the EVs, whereas immunocytochemistry indicated that the EVs most likely derived from the astrocytes and the neurons in the culture. The identified A beta-induced sorting of apoE into EVs from cultured neuroglial cells suggests a possible role for intercellular transfer of apoE in AD pathology and encourage future studies to fully elucidate the clinical relevance of this event.

Keywords
Alzheimer’s disease, amyloid-beta, apolipoprotein E, astrocytes, exosomes, extracellular vesicles, mass spectrometry, neurons, shedding microvesicles
National Category
Medical and Health Sciences Neurosciences
Identifiers
urn:nbn:se:uu:diva-331137 (URN)10.3233/JAD-170278 (DOI)000408582800025 ()
Funder
Swedish Research CouncilStiftelsen Gamla TjänarinnorÅke Wiberg Foundation
Available from: 2017-10-11 Created: 2017-10-11 Last updated: 2018-01-13Bibliographically approved
3. The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death.
Open this publication in new window or tab >>The Aβ protofibril selective antibody mAb158 prevents accumulation of Aβ in astrocytes and rescues neurons from Aβ-induced cell death.
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(English)In: Article in journal (Refereed) Submitted
Keywords
Alzheimer’s disease, amyloid-β, antibody, clearance, astrocyte, neuron
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-264223 (URN)
Available from: 2015-10-15 Created: 2015-10-07 Last updated: 2017-11-28
4. Extracellular vesicle-mediated amyloid-β toxicity in cortical neurons
Open this publication in new window or tab >>Extracellular vesicle-mediated amyloid-β toxicity in cortical neurons
(English)Manuscript (preprint) (Other academic)
Keywords
Alzheimer’s disease, amyloid-β, extracellular vesicles, apoptosis, neurons, synapses, mitochondria
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-334346 (URN)
Available from: 2017-11-22 Created: 2017-11-22 Last updated: 2017-11-28

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