Numerous studies point to an important role for astrocytes in Alzheimer’s disease (AD). Astrocytes are a type of glial cell that plays multiple roles in supporting a healthy brain. One of their most well-known functions is as an innate immune cell that mounts an inflammatory response to brain injuries, cell debris or other toxic materials.
Early in AD, when plaques begin accumulating, astrocytes become reactive. These reactive astrocytes might be critical mediators of how amyloid triggers tau pathology. Despite more attention being paid to this interesting cell type, there is still a lot to be learned about how astrocytes contribute to the onset or progression of AD. Identifying the molecules that astrocytes use to influence other cell types and contribute to amyloid- or tau-related pathologies could lead to novel drug targets.
Dr. Gallardo’s previous research identified a key protein in astrocytes, called NKA (abbreviation of Na+/K+ ATPase), as a critical regulator of neuron death in a mouse model of amyotrophic lateral sclerosis (ALS). To determine if NKA might also play a role in AD, Dr. Gallardo’s lab tested the impact of a pharmacological NKA inhibitor in a tau mouse model (PS19) and found that blocking NKA reduced astrocyte reactivity, lowered the levels of inflammatory molecules (cytokines) and protected against tau pathology.
When NKA is activated, it uses a lot of ATP (the cell’s energy supply). A sensor molecule called AMPK detects this increased demand and activates a program to help replenish ATP levels in the cell. Dr. Gallardo found that AMPK activity was increased in both human AD and amyloid beta mouse models (APP/PS1). In this proposal, his team will further explore the role of NKA and AMPK in AD pathogenesis. They hypothesized that, under AD conditions, astrocytes increase NKA activity, which triggers AMPK signaling and promotes the reactive astrocyte state. This cascade of events leads to neuroinflammation and related amyloid pathologies.
The Gallardo lab proposed three experimental aims to test this hypothesis. In the first aim, they are testing the impact of modulating NKA activity on cellular ATP levels and AMPK activation. They are using genetic and pharmacological methods to block or reduce NKA in reactive astrocytes in cell culture and in APP/PS1 mice. In the second aim, they are evaluating the impact of AMPK on amyloid pathology. They are employing two approaches to knock out AMPK in astrocytes of APP/PS1 mice and are measuring outcomes related to inflammation, amyloid beta pathology and cognitive performance. In the final aim, their goal is to identify the downstream signaling partners directly activated by AMPK that cause astrocytes to become reactive. They are using two complementary screens for this search: (1) evaluating a pre-selected set of candidates and (2) using an unbiased approach. In the first screen, they are measuring activity and other biochemical changes in candidate molecules known to regulate mitochondrial respiration—the process within mitochondria that breaks down glucose with the help of oxygen to produce ATP. In parallel, they are performing an unbiased screen by tagging all proteins that AMPK acts on, then using mass spectroscopy to identify those proteins. These experiments are being performed in amyloid mice and astrocytes in culture.
In the first year of funding, Dr. Gallardo’s team made substantial progress on the proposed work. They identified a novel pathway dictating astrocyte responses to ATP levels that differs between reactive and non-reactive astrocytes. In both, they observed an increase in AMPK activity, but that activity triggered different downstream effects in each. As part of the second aim, they further found that AMPK isoforms might be regulating these different downstream effects in astrocytes, which they aim to test by genetic knockout in cultured astrocytes and in APP/PS1 mice. In the next year of funding, they plan to complete the evaluation of these genetic AMPK knockouts and identify downstream signaling partners activated by AMPK. Overall, this project is shedding light on key aspects of the signaling pathways underlying the reactivity of astrocytes and is helping inform future therapeutic approaches for AD.
