Final Report Summary - SAVING DYING NEURONS (Immune responses in neurodegenerative diseases: Protection or progression?)
Many neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases, are progressive and incurable. It is clear that immune and inflammatory responses are involved in these diseases, but in general it is unclear whether they are protective or promote the disease process. The overall aim of the proposed research is gaining insight into contribution of immune responses to neurodegeneration in vivo. The specific objectives are:
• Define what the key immunological events are and when they occur during degeneration.
• Elucidate how different non-immunogenic or inflammatory responses contribute to disease progression.
• Find small molecules that interfere with the immune response to modulate disease progression.
AIM 1 was executed as planned, has been successfully completed, and a manuscript based on this work was published in 2014 (van Ham et al.). Using our new model we characterised the exact types of immune cells that play a role in neurodegeneration and the ones that are not involved. Microglia, the macrophages of the brain, are the main immune cells involved. Furthermore, we show that two types of cells are involved and they exhibit different temporal dynamics. Additionally, we find important new insight related to the resolution of the immune response during recovery from neurodegeneration (neuroregeneration). We show this immune response in the brain is resolved in a way similar to wound healing, involving the apoptosis of phagocytic immune cells. The publication was included in a special issue on zebrafish models and translational medicine (http://dmm.biologists.org/content/7/7/731). Additionally, together with one of the PhD students in my lab we wrote a review on the use of zebrafish in neuroimmunology, including our latest published data, and how this may contribute to gain insight in the role of the immune system in –preventing- brain disease. This review (Oosterhof, Boddeke and van Ham, 2015) was published in the May issue of main journal on glial cells (GLIA) and included as the cover image (http://onlinelibrary.wiley.com/doi/10.1002/glia.22780/abstract).
Regarding AIM 2, we have performed RNA sequencing on microglia to identify genes controlling immune responses. This is a deviation from the original plan. We successfully isolated microglia from zebrafish brains (controls versus neurodegeneration fish), purified these using flow cytometry/FACS, and subsequently isolated RNA, and performed whole RNA sequencing. Interestingly, we discovered that the zebrafish microglial transcriptome is highly conserved with mammals, showing that fish brain immune cells are very similar to mammalian brain immune cells. Additionally, we identified genes expression of which is increased in microglia during neurodegeneration, showing microglia unexpectedly become highly proliferative. We confirmed this using in vivo microscopy, and are currently performing experiments to understand the genetic & molecular mechanisms controlling this proliferative activation response. These results represent a first analysis of genome-wide gene expression in zebrafish microglia under healthy and neurodegeneration conditions and a manuscript based on these pioneering experiments was published in the journal GLIA (Oosterhof ... van Ham, GLIA; see publications). Recent developments in genome editing, mainly by the CRISPR/Cas9 system, finally allow us to effectively knockout genes in zebrafish and we are currently taking full advantage of this to study the new genetic mechanisms we identified using RNA sequencing. These reverse genetics experiments largely replace the small molecule targeting approach in AIM 3. Manuscripts based on this work are in preparation. In all this work has led to new insights in the role of immune cells, mainly microglia, in recovery from neuronal injury, elucidation of the zebrafish microglia transcriptome in the healthy and neurodegenerative brain, and its conservation with mammals. These results particularly pave the way for detailed investigations of microglial genetic mechanisms in health and disease using the zebrafish as a model system.
• Define what the key immunological events are and when they occur during degeneration.
• Elucidate how different non-immunogenic or inflammatory responses contribute to disease progression.
• Find small molecules that interfere with the immune response to modulate disease progression.
AIM 1 was executed as planned, has been successfully completed, and a manuscript based on this work was published in 2014 (van Ham et al.). Using our new model we characterised the exact types of immune cells that play a role in neurodegeneration and the ones that are not involved. Microglia, the macrophages of the brain, are the main immune cells involved. Furthermore, we show that two types of cells are involved and they exhibit different temporal dynamics. Additionally, we find important new insight related to the resolution of the immune response during recovery from neurodegeneration (neuroregeneration). We show this immune response in the brain is resolved in a way similar to wound healing, involving the apoptosis of phagocytic immune cells. The publication was included in a special issue on zebrafish models and translational medicine (http://dmm.biologists.org/content/7/7/731). Additionally, together with one of the PhD students in my lab we wrote a review on the use of zebrafish in neuroimmunology, including our latest published data, and how this may contribute to gain insight in the role of the immune system in –preventing- brain disease. This review (Oosterhof, Boddeke and van Ham, 2015) was published in the May issue of main journal on glial cells (GLIA) and included as the cover image (http://onlinelibrary.wiley.com/doi/10.1002/glia.22780/abstract).
Regarding AIM 2, we have performed RNA sequencing on microglia to identify genes controlling immune responses. This is a deviation from the original plan. We successfully isolated microglia from zebrafish brains (controls versus neurodegeneration fish), purified these using flow cytometry/FACS, and subsequently isolated RNA, and performed whole RNA sequencing. Interestingly, we discovered that the zebrafish microglial transcriptome is highly conserved with mammals, showing that fish brain immune cells are very similar to mammalian brain immune cells. Additionally, we identified genes expression of which is increased in microglia during neurodegeneration, showing microglia unexpectedly become highly proliferative. We confirmed this using in vivo microscopy, and are currently performing experiments to understand the genetic & molecular mechanisms controlling this proliferative activation response. These results represent a first analysis of genome-wide gene expression in zebrafish microglia under healthy and neurodegeneration conditions and a manuscript based on these pioneering experiments was published in the journal GLIA (Oosterhof ... van Ham, GLIA; see publications). Recent developments in genome editing, mainly by the CRISPR/Cas9 system, finally allow us to effectively knockout genes in zebrafish and we are currently taking full advantage of this to study the new genetic mechanisms we identified using RNA sequencing. These reverse genetics experiments largely replace the small molecule targeting approach in AIM 3. Manuscripts based on this work are in preparation. In all this work has led to new insights in the role of immune cells, mainly microglia, in recovery from neuronal injury, elucidation of the zebrafish microglia transcriptome in the healthy and neurodegenerative brain, and its conservation with mammals. These results particularly pave the way for detailed investigations of microglial genetic mechanisms in health and disease using the zebrafish as a model system.