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Project
Molecular imaging of neurodegeneration and neurogenesis in animal models.
With more than thousand disorders of the brain and nervous system, affecting up to one billion people worldwide, the importance of neuroscienceresearch cannot be overstated (1). The development of new technologies is indispensable to expand our knowledge of the molecular processes occurring in the healthy and diseased brain. Bioluminescence imaging (BLI) is a simple, relatively inexpensive and sensitive imaging technology thatallows non-invasive evaluation of a molecular process in the same groupof animals over time. The main objectives of this thesis were to develop and characterize new tools to follow up different neurological processes in vivo with BLI.
Alpha-synuclein (aSYN) aggregation plays a pivotal role in the pathogenesis of Parkinsons disease and other synucleinopathies. In this multi-step process, oligomerization of aSYN monomers is the first step in the formation of fibrils and intracytoplasmic inclusions that are detected in the brain of patients. Although the aSYN oligomers are generally considered to be the culprit of these diseases, the methodology currently available to follow up oligomerization in cells and in brain is inadequate. For this reason, we designed a bioluminescent protein complementation assay, whereby firefly luciferase (Fluc) is split and its N-terminal and C-terminal parts are fused to aSYN. In cell culture, aSYN oligomerization resulted in successful luciferase complementation and consequently in the production of visible light. In addition, the Fluc tags did not appear to influence the aggregation properties of aSYN. Next, aSYN oligomerization could be non-invasively monitoredwith BLI in the mouse striatum and substantia nigra up to 8 months postinjection. Importantly, the visualized aSYN oligomers retained their toxic and aggregation properties in vivo. Finally, the effect of FK506, known to inhibit aSYN fibril formation was investigated. We showed that FK506 inhibited the observed aSYN oligomerization both in cell culture andin mouse brain. We believe that this bioluminescent complementation assay will increase our insight in the role of aSYN oligomers in synucleinopathies and will open new opportunities to evaluate potential aSYN-basedneuroprotective therapies in cell culture and in mouse brain.
Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone (SVZ) and the dentate gyrus (DG). Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Stereotactic injection of lentiviral (LV) vectors encoding Fluc into the SVZ previously allowedus to monitor the migration of neural stem cell (NSC) progeny with BLI.However, LV vectors not only transduce NSCs but also neighboring astrocytes and mature neurons. This results in a high BLI signal emerging fromthe SVZ that interferes with the measurement of the migrating cells. Inthe second part of this thesis, we developed conditional Cre-Flex LV vectors that allowed restrictive expression of Fluc and eGFP in NSCs afterinjection in the SVZ of transgenic Nestin-Cre mice. Stroke is known to affect adult neurogenesis, but the exact origin of the migrating cells and their differentiation potential is controversial. For this reason, weapplied the Cre-Flex LV vectors to monitor the neurogenic response in astroke model. After labeling the NSCs of the SVZ with the Cre-Flex LV vectors, induction of a stroke resulted in a transient increase in both the BLI signal and the number of eGFP positive cells. In addition, a clear migration of the NSC progeny towards the stroke region was detected with BLI. Histological analysis revealed both astrocytic and neurogenic differentiation of the NSC progeny in the peri-infarct region. This new approach thus allows non-invasive and specific monitoring of NSCs and their progeny over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.
Although tremendous progress has been made in our knowledge of adult neurogenesis in the other neurogenic region, the DG, important questions remain regarding the identity and the behavior of NSCs in the DG. In the third part of this thesis we therefore aimed to apply the Cre-Flex LV vectors to study neurogenesis in the DG with BLI and histology. Stereotactic injection of the Cre-Flex LV vectors into the DG of Nestin-Cre mice resulted in specific labeling of thenestin positive NSCs. The labeled cell population could be detected with BLI until 9 months post injection, but due to technical limitations, the continuous increase in the number of eGFP-labeled cells was not observed with BLI. Nevertheless, we took advantage of the specific labeling of the nestin positive NSCs to analyze their neurogenic potential by performing extensive histological analysis at different time points. This long-term fate mapping revealed that a stable pool of labeled nestin positive NSCs continuously contributed to the generation of newborn neurons until 9 months post injection. Therefore, we believe that this approach will help to address the remaining questions regarding NSC identity and behavior in the DG.
Alpha-synuclein (aSYN) aggregation plays a pivotal role in the pathogenesis of Parkinsons disease and other synucleinopathies. In this multi-step process, oligomerization of aSYN monomers is the first step in the formation of fibrils and intracytoplasmic inclusions that are detected in the brain of patients. Although the aSYN oligomers are generally considered to be the culprit of these diseases, the methodology currently available to follow up oligomerization in cells and in brain is inadequate. For this reason, we designed a bioluminescent protein complementation assay, whereby firefly luciferase (Fluc) is split and its N-terminal and C-terminal parts are fused to aSYN. In cell culture, aSYN oligomerization resulted in successful luciferase complementation and consequently in the production of visible light. In addition, the Fluc tags did not appear to influence the aggregation properties of aSYN. Next, aSYN oligomerization could be non-invasively monitoredwith BLI in the mouse striatum and substantia nigra up to 8 months postinjection. Importantly, the visualized aSYN oligomers retained their toxic and aggregation properties in vivo. Finally, the effect of FK506, known to inhibit aSYN fibril formation was investigated. We showed that FK506 inhibited the observed aSYN oligomerization both in cell culture andin mouse brain. We believe that this bioluminescent complementation assay will increase our insight in the role of aSYN oligomers in synucleinopathies and will open new opportunities to evaluate potential aSYN-basedneuroprotective therapies in cell culture and in mouse brain.
Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone (SVZ) and the dentate gyrus (DG). Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Stereotactic injection of lentiviral (LV) vectors encoding Fluc into the SVZ previously allowedus to monitor the migration of neural stem cell (NSC) progeny with BLI.However, LV vectors not only transduce NSCs but also neighboring astrocytes and mature neurons. This results in a high BLI signal emerging fromthe SVZ that interferes with the measurement of the migrating cells. Inthe second part of this thesis, we developed conditional Cre-Flex LV vectors that allowed restrictive expression of Fluc and eGFP in NSCs afterinjection in the SVZ of transgenic Nestin-Cre mice. Stroke is known to affect adult neurogenesis, but the exact origin of the migrating cells and their differentiation potential is controversial. For this reason, weapplied the Cre-Flex LV vectors to monitor the neurogenic response in astroke model. After labeling the NSCs of the SVZ with the Cre-Flex LV vectors, induction of a stroke resulted in a transient increase in both the BLI signal and the number of eGFP positive cells. In addition, a clear migration of the NSC progeny towards the stroke region was detected with BLI. Histological analysis revealed both astrocytic and neurogenic differentiation of the NSC progeny in the peri-infarct region. This new approach thus allows non-invasive and specific monitoring of NSCs and their progeny over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.
Although tremendous progress has been made in our knowledge of adult neurogenesis in the other neurogenic region, the DG, important questions remain regarding the identity and the behavior of NSCs in the DG. In the third part of this thesis we therefore aimed to apply the Cre-Flex LV vectors to study neurogenesis in the DG with BLI and histology. Stereotactic injection of the Cre-Flex LV vectors into the DG of Nestin-Cre mice resulted in specific labeling of thenestin positive NSCs. The labeled cell population could be detected with BLI until 9 months post injection, but due to technical limitations, the continuous increase in the number of eGFP-labeled cells was not observed with BLI. Nevertheless, we took advantage of the specific labeling of the nestin positive NSCs to analyze their neurogenic potential by performing extensive histological analysis at different time points. This long-term fate mapping revealed that a stable pool of labeled nestin positive NSCs continuously contributed to the generation of newborn neurons until 9 months post injection. Therefore, we believe that this approach will help to address the remaining questions regarding NSC identity and behavior in the DG.
Date:1 Oct 2009 → 24 Jun 2014
Keywords:Molecular imaging
Disciplines:Neurosciences, Biological and physiological psychology, Cognitive science and intelligent systems, Developmental psychology and ageing
Project type:PhD project