In Vivo Monitoring and Phenotyping of Tau Aggregation
MentorBradley Hyman, MD, PhD Massachusetts General Hospital, Harvard Medical School
A major driver of Alzheimer’s disease (AD) is the accumulation of the protein tau that travels through the human brain in a constant pattern. Tau molecules become misshapen and aggregate in AD, though no one has yet identified how, or even if, these tau accumulations result in neuronal death. In this research, we have developed a fluorescent tool that will allow us to watch tau collect in neurons both in cell culture as well as the living adult mouse brain. Using this tool, this research aims to observe directly, in real time, what happens once a neuron develops a tau aggregate, as well as to study which genes increase or decrease in a neuron once it develops one of these tau accumulations. Together, these data will help us better understand the immediate changes that occur in adult neurons when they develop AD-like tau accumulations and may help identify new druggable pathways involved in the development of AD in human patients.
Note: This grant was terminated by the investigator in February of 2018 when she left Harvard University for an industry position.
The overall goal of this project is to help identify possible mechanisms that are involved in tau-mediated neuronal loss that may then serve as the basis for developing new therapeutic targets for AD.
In the first aim of this study, I will use a virus that we have termed TauFRET2 that expresses the aggregation-prone repeat domain of the protein tau fused with both cyan fluorescent protein (CFP) and yellow florescent protein (YFP). I will first apply the virus to primary neurons from the tauopathy mouse model PS19 to monitor the spatial localization of tau aggregates in real time using time lapse imaging, as well as sort out those neurons that spontaneously develop tau aggregates to determine aggregation kinetics. Additionally, I will inject the TauFRET2 virus into the cortex of adult tauopathy mice. This will enable us to monitor tau aggregate formation (and possible degradation) in vivo in order to study whether the formation of tau aggregates directly leads to neuronal death. In the second aim, the goal is both to quantify the tau aggregates in vivo using flow cytometry, as well as sort out those aggregate positive neurons from the adult mouse brain and send for RNA-Seq analysis. The quantification of aggregates in vivo will give us a sense of aggregation dynamics in the living brain. The subsequent gene expression analysis will then give us a window into the downstream molecular pathways that are up and down-regulated upon the formation of a tau aggregate.
In AD, there are several models for monitoring how tau aggregates spread from cell to cell. What is lacking, however, is a tool that provides sensitive detection of tau aggregation and spreading in animal models in real time, enabling the longitudinal visualization and isolation of tau aggregation and potential subsequent toxicity. This is where the current study comes in. We have developed such a tool - TauFRET2 - that will allow us to fill in this gap in the field and allow us to visualize tau aggregates in the living adult mouse brain over time as well as isolate those neurons that contain tau aggregates for gene expression analysis. By combining this novel TauFRET2 system with several downstream techniques to image, quantify, and isolate tau aggregate-containing neurons from adult mice, this innovate study will help tease apart some of the basic, but crucially important, questions surrounding neuronal toxicity that is mediated by tau aggregation.
Upon completion of this work, the proposed studies will shed light on the dynamics of tau aggregation and allow us isolate tau aggregate-containing neurons to explore gene expression changes following tau aggregate formation. These results may serve as the basis for screening already-developed drugs to determine if they can prevent the formation of additional aggregates in vitro and in vivo, and for identifying new molecular pathways that are up or down-regulated in the presence of tau aggregation that in turn can be used to find novel AD therapeutic targets.