Roles of compromised mitophagy in Alzheimer’s disease
The purpose of this project is to test a novel hypothesis that a defective mitophagy contributes to the aetiology and progression of Alzheimer’s disease (AD). For this purpose, we will employ several mouse models in the study, including a wild type (WT) C57BL/6, a mitophagy over-expressed strain (Ulk-1 overexpressed strain), and three transgenic mouse models of AD including: APP/PS1 (Scheffler et al., 2011), amyloid (5×FAD (Oakley et al., 2006)), and tau (P301S (Allen et al., 2002)).
We will use these mouse models to investigate the mitochondrial function and levels of mitophagy in relation to AD-associated behavioural and pathological phenotypes presented in these mouse models. Subsequently, we will evaluate whether upregulation of mitophagy, both pharmacologically (e.g., NAD+ precursors, actinonin, and urolithin A, UA (Ryu et al., 2016)) and genetically (e.g., Ulk1 overexpression, novel model), can inhibit cognitive decline and reduces amyloid and tau pathologies.
Based on experience from previous experiments and published works by others, we do not foresee any adverse effect. The design of the current study is modelled on our experiments conducted at the Laboratory of Molecular Gerontology (LMG), National Institute on Aging (NIA), USA. All these experiments were performed and with ethical approval at LMG, NIA, USA whereby Fang EF was a postdoc there during 2012-2017. Consistently, there was no detectable side effect of UA (Ryu et al., 2016) (and personal communication) or NAD+ precursors (Sorrentino et al., 2017) in the mouse studies in another researcher’s laboratory. Thus, we will use the aforementioned doses for the pharmacological treatments.
Recent evidence has shown that Alzheimer's and sleep are closely interconnected. However, the mechanism of this intervention is not well known. In particular, the connection between mitophagy and sleep has not been a well-explored field yet. For this reason, we would like to include a behavioral test related to the sleep pattern and its relationship with mitophagy In our experiments. As well, we want to provide functional tests on how mitophagy inducers can control neuronal activity and its relationship with sleep .
To reach these aims we plan to do AAV to down-regulate or over-express a transcription factor called REST that has been reported to connect mitophagy and synapse function. In order to assess this, we want/need to conduct AAV injections on the mice and to use microPET to evaluate synapse and mitophagy markers in different areas of the brain.
In all in vivo tests, the number of mice in each group will be the minimum required to allow valid statistical analysis. Based on our previous experience as well as studies from colleagues in the AD fields (Fang et al., 2016; Sorrentino et al., 2017), we propose to use 10 mice/ experimental group (5 male and 5 female) at each stage (age group). In total, we will require 360 mice. To further reduce the numbers of animals used and to refine animal use wherever possible, animals may be retested within the same protocol for assessment of locomotor and cognitive status. We will also reduce the use of primary mouse neuronal cells (freshly prepared from culling the pups) by using induced pluripotent stem cells (iPSCs). The Fang group is establishing this technique in the Akershus University Hospital. Furthermore, we have already replaced some of the mouse experiments by using the small roundworm C. elegans.In summary, in these proposed set of experiments, we aim to question whether upregulation of mitophagy can reduce or inhibit the development and/or progression of AD behavioural and pathological features. There are over 44.5 million people suffered with AD, thus the success of this project will have enormous benefit to the AD patients as well to the healthcare system in Norway and across the globe.
We will use these mouse models to investigate the mitochondrial function and levels of mitophagy in relation to AD-associated behavioural and pathological phenotypes presented in these mouse models. Subsequently, we will evaluate whether upregulation of mitophagy, both pharmacologically (e.g., NAD+ precursors, actinonin, and urolithin A, UA (Ryu et al., 2016)) and genetically (e.g., Ulk1 overexpression, novel model), can inhibit cognitive decline and reduces amyloid and tau pathologies.
Based on experience from previous experiments and published works by others, we do not foresee any adverse effect. The design of the current study is modelled on our experiments conducted at the Laboratory of Molecular Gerontology (LMG), National Institute on Aging (NIA), USA. All these experiments were performed and with ethical approval at LMG, NIA, USA whereby Fang EF was a postdoc there during 2012-2017. Consistently, there was no detectable side effect of UA (Ryu et al., 2016) (and personal communication) or NAD+ precursors (Sorrentino et al., 2017) in the mouse studies in another researcher’s laboratory. Thus, we will use the aforementioned doses for the pharmacological treatments.
Recent evidence has shown that Alzheimer's and sleep are closely interconnected. However, the mechanism of this intervention is not well known. In particular, the connection between mitophagy and sleep has not been a well-explored field yet. For this reason, we would like to include a behavioral test related to the sleep pattern and its relationship with mitophagy In our experiments. As well, we want to provide functional tests on how mitophagy inducers can control neuronal activity and its relationship with sleep .
To reach these aims we plan to do AAV to down-regulate or over-express a transcription factor called REST that has been reported to connect mitophagy and synapse function. In order to assess this, we want/need to conduct AAV injections on the mice and to use microPET to evaluate synapse and mitophagy markers in different areas of the brain.
In all in vivo tests, the number of mice in each group will be the minimum required to allow valid statistical analysis. Based on our previous experience as well as studies from colleagues in the AD fields (Fang et al., 2016; Sorrentino et al., 2017), we propose to use 10 mice/ experimental group (5 male and 5 female) at each stage (age group). In total, we will require 360 mice. To further reduce the numbers of animals used and to refine animal use wherever possible, animals may be retested within the same protocol for assessment of locomotor and cognitive status. We will also reduce the use of primary mouse neuronal cells (freshly prepared from culling the pups) by using induced pluripotent stem cells (iPSCs). The Fang group is establishing this technique in the Akershus University Hospital. Furthermore, we have already replaced some of the mouse experiments by using the small roundworm C. elegans.In summary, in these proposed set of experiments, we aim to question whether upregulation of mitophagy can reduce or inhibit the development and/or progression of AD behavioural and pathological features. There are over 44.5 million people suffered with AD, thus the success of this project will have enormous benefit to the AD patients as well to the healthcare system in Norway and across the globe.