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Cellular and Molecular Responses to Traumatic Brain Injury
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients.

The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells.

The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 966
Keyword [en]
Traumatic Brain Injury, Astrocyte, Apoptosis, Biomarkers, Ezrin, Actin, Extracellular Proteins, Degradation, Lysosome, Antigen Presentation
National Category
Neurosciences
Research subject
Neuroscience; Neurosurgery
Identifiers
URN: urn:nbn:se:uu:diva-215154ISBN: 978-91-554-8845-1 (print)OAI: oai:DiVA.org:uu-215154DiVA: diva2:686360
Public defence
2014-02-28, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-02-06 Created: 2014-01-11 Last updated: 2014-02-10
List of papers
1. Engulfing Astrocytes Protect Neurons from Contact-Induced Apoptosis following Injury
Open this publication in new window or tab >>Engulfing Astrocytes Protect Neurons from Contact-Induced Apoptosis following Injury
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, e33090- p.Article in journal (Refereed) Published
Abstract [en]

Clearing of dead cells is a fundamental process to limit tissue damage following brain injury. Engulfment has classically been believed to be performed by professional phagocytes, but recent data show that non-professional phagocytes are highly involved in the removal of cell corpses in various situations. The role of astrocytes in cell clearance following trauma has however not been studied in detail. We have found that astrocytes actively collect and engulf whole dead cells in an in vitro model of brain injury and thereby protect healthy neurons from bystander cell death. Time-lapse experiments showed that migrating neurons that come in contact with free-floating cell corpses induced apoptosis, while neurons that migrate through groups of dead cells, garnered by astrocytes, remain unaffected. Furthermore, apoptotic cells are present within astrocytes in the mouse brain following traumatic brain injury (TBI), indicating a possible role for astrocytes in engulfment of apoptotic cells in vivo. qRT-PCR analysis showed that members of both ced pathways and Megf8 are expressed in the cell culture, indicating their possible involvement in astrocytic engulfment. Moreover, addition of dead cells had a positive effect on the protein expression of MEGF10, an ortholog to CED1, known to initiate phagocytosis by binding to phosphatidylserine. Although cultured astrocytes have an immense capacity for engulfment, seemingly without adverse effects, the ingested material is stored rather than degraded. This finding might explain the multinuclear astrocytes that are found at the lesion site in patients with various brain disorders.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-175860 (URN)10.1371/journal.pone.0033090 (DOI)000304118900009 ()
Available from: 2012-06-13 Created: 2012-06-13 Last updated: 2017-12-07Bibliographically approved
2. Slow degradation in phagocytic astrocytes can be enhanced by lysosomal acidification
Open this publication in new window or tab >>Slow degradation in phagocytic astrocytes can be enhanced by lysosomal acidification
2015 (English)In: Glia, ISSN 0894-1491, E-ISSN 1098-1136, Vol. 63, no 11, 1997-2009 p.Article in journal (Refereed) Published
Abstract [en]

Inefficient lysosomal degradation is central in the development of various brain disorders, but the underlying mechanisms and the involvement of different cell types remains elusive. We have previously shown that astrocytes effectively engulf dead cells, but then store, rather than degrade the ingested material. In the present study we identify reasons for the slow digestion and ways to accelerate degradation in primary astrocytes. Our results show that actin-rings surround the phagosomes for long periods of time, which physically inhibit the phago-lysosome fusion. Furthermore, astrocytes express high levels of Rab27a, a protein known to reduce the acidity of lysosomes by Nox2 recruitment, in order to preserve antigens for presentation. We found that Nox2 colocalizes with the ingested material, indicating that it may influence antigen processing also in astrocytes, as they express MHC class II. By inducing long-time acidification of astrocytic lysosomes using acidic nanoparticles, we could increase the digestion of astrocyte-ingested, dead cells. The degradation was, however, normalized over time, indicating that inhibitory pathways are up-regulated in response to the enhanced acidification.

Keyword
Phagocytosis, in vitro model, digestion, antigen presentation, Rab27a, Nox2, pHrodo, actin, Latrunculin
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-215150 (URN)10.1002/glia.22873 (DOI)000361185000008 ()
Funder
Magnus Bergvall Foundation
Note

Manuscript title: Degradation of Ingested Dead Cells in Phagocytic Astrocytes is Tightly Regulated, but can be Enhanced by Lysosomal Acidifcatio

Available from: 2014-01-11 Created: 2014-01-11 Last updated: 2017-12-06Bibliographically approved
3. Identification of Injury Specific Proteins in a Cell Culture Model of Traumatic Brain Injury
Open this publication in new window or tab >>Identification of Injury Specific Proteins in a Cell Culture Model of Traumatic Brain Injury
Show others...
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e55983- p.Article in journal (Refereed) Published
Abstract [en]

The complicated secondary molecular and cellular mechanisms following traumatic brain injury (TBI) are still not fully understood. In the present study, we have used mass spectrometry to identify injury specific proteins in an in vitro model of TBI. A standardized injury was induced by scalpel cuts through a mixed cell culture of astrocytes, oligodendrocytes and neurons. Twenty-four hours after the injury, cell culture medium and whole-cell fractions were collected for analysis. We found 53 medium proteins and 46 cell fraction proteins that were specifically expressed after injury and the known function of these proteins was elucidated by an extensive literature survey. By using time-lapse microscopy and immunostainings we could link a large proportion of the proteins to specific cellular processes that occur in response to trauma; including cell death, proliferation, lamellipodia formation, axonal regeneration, actin remodeling, migration and inflammation. A high percentage of the proteins uniquely expressed in the medium after injury were actin-related proteins, which normally are situated intracellularly. We show that two of these, ezrin and moesin, are expressed by astrocytes both in the cell culture model and in mouse brain subjected to experimental TBI. Interestingly, we found many inflammation-related proteins, despite the fact that cells were present in the culture. This study contributes with important knowledge about the cellular responses after trauma and identifies several potential cell-specific biomarkers.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-197400 (URN)10.1371/journal.pone.0055983 (DOI)000315157200097 ()
Available from: 2013-04-01 Created: 2013-03-25 Last updated: 2017-12-06Bibliographically approved
4. Extracellular Ezrin - a Novel Biomarker for Traumatic Brain Injury
Open this publication in new window or tab >>Extracellular Ezrin - a Novel Biomarker for Traumatic Brain Injury
Show others...
2015 (English)In: Journal of Neurotrauma, ISSN 0897-7151, E-ISSN 1557-9042, Vol. 32, no 4, 244-251 p.Article in journal (Refereed) Published
Abstract [en]

Traumatic brain injury (TBI) is a heterogeneous disease, and the discovery of diagnostic and prognostic TBI biomarkers is highly desirable in order to individualize patient care. We have previously published a study in which we identified possible TBI biomarkers by mass spectrometry 24 h after injury in a cell culture model. Ezrin-radixin-moesin (ERM) proteins were found abundantly in the medium after trauma, and in the present study we have identified extracellular ezrin as a possible biomarker for brain trauma by analyzing cell culture medium from injured primary neurons and glia and by measuring ezrin in cerebrospinal fluid (CSF) from both rats and humans. Our results show that extracellular ezrin concentration was substantially increased in cell culture medium after injury, but that the intracellular expression of the protein remained stable over time. Controlled cortical impact injured rats showed an increased amount of ezrin in CSF at both day 3 and day 7 after trauma. Moreover, ezrin was present in all ventricular CSF samples from seven humans with severe TBI. In contrast to intracellular ezrin, which is distinctly activated following TBI, extracellular ezrin is nonphosphorylated. This is the first report of extracellular ERM proteins in human and experimental models of TBI, providing a scientific foundation for further assessment of ezrin as a potential biomarker.

Keyword
TBI, Moesin, Radixin, pERM, ERM, actin, extracellular proteins, astrocytes
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-215151 (URN)10.1089/neu.2014.3517 (DOI)000349314900005 ()25087457 (PubMedID)
Available from: 2014-01-11 Created: 2014-01-11 Last updated: 2017-12-06Bibliographically approved

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