Substantial genetic and experimental evidence implicates microglia in Alzheimer’s disease (AD). As the brain’s resident immune cells, microglia respond to neuronal injury or pathogens by shifting from a homeostatic, surveillance state to an activated state. When activated, they can increase inflammatory signaling or begin to engulf and clear foreign material or cellular debris, such as damaged neuronal synapses, through a process called phagocytosis. These activated states are an important part of how microglia maintain a healthy brain environment, but they can become damaging if left unchecked for too long. However, researchers do not fully understand the signals that regulate these processes or what impact this has on amyloid pathology and cognitive function. Identifying specific signaling pathways that regulate microglia functions impacted by AD is expected to provide novel targets for future drug development efforts.
Dr. Bilbo is investigating the role of the cytokine signaling molecule IL34 (interleukin-34) in AD. Interestingly, IL34 is produced by neurons—not microglia—and binds to a receptor on the microglial surface. A genetic variant of IL34 has been linked to Alzheimer’s, and both mouse models and human AD brains show reduced IL34 levels, making it a gene of interest. Dr. Bilbo’s prior work in brain development showed that IL34 levels rise just as microglia begin to reduce their synaptic pruning activity. During normal development, microglia help refine the brain by removing weaker or unnecessary brain connections. This suggests that IL34 may act as a brake to limit excessive microglial phagocytosis.
In the last funding cycle, the Bilbo team tested the idea that IL34 signaling returns microglia to a homeostatic, non-phagocytic state. They hypothesized that lower IL34 levels lock microglia in an overactive state. While this initially ensures that microglia clear amyloid, it ultimately leads to synapse loss and cognitive decline because the microglia cannot return to their resting state.
Preliminary results support their idea: in young 5xFAD mice with reduced IL34 levels, there were smaller and fewer plaques, especially in males, without increased microglial synapse engulfment at early stages. They also observed that IL34 loss accelerated motor and anxiety deficits in male mice. After mapping the distribution of amyloid in the brain, they discovered that the lateral septum, a region involved in anxiety, had the highest plaque burden, rather than the hippocampus, their original focus. These findings suggest IL34’s influence on microglia, amyloid, and behavior may extend beyond the hippocampus. In this follow-on project, Dr. Bilbo is expanding her hypothesis to include the lateral septum and exploring how IL34 affects motor and anxiety-related behaviors alongside microglial states and functions in AD.
The Bilbo team has proposed two aims to test their hypothesis. In the first aim, they are testing whether increasing IL34 in the lateral septum prevents motor and anxiety deficits as well as cellular pathologies. The team will use 5xFAD mice and put them through a battery of behavioral tests before and after increasing IL34 levels. To increase IL34 levels, they will inject modified viruses that deliver IL34-producing genes directly to brain cells in the lateral septum. They will also quantify microglia numbers, amyloid load, microglial engulfment, and synapses in mice with and without increased IL34. In the second aim, they are testing whether increasing IL34 levels switches microglia from a disease-associated state to a homeostatic, resting one. 5xFAD mice will be bred with another mouse line that allows for specific isolation of disease-associated microglia (Clec7a-CreER mice). IL34 levels will be increased, just as in Aim 1, at two ages: before and after plaque accumulation. The effects of IL34 will be assessed by measuring microglia numbers and other indicators of microglial function and activation state using advanced genetic sequencing (scRNA-seq) and tissue analysis techniques.
At the end of the first year of funding, the team has made strong progress on both aims. For Aim 1, they tested whether boosting IL34 in the lateral septum improved behavior and protected brain cells in Alzheimer’s model mice. They demonstrated that their method for increasing IL34 levels in the lateral septum was effective, and that it also increased levels of the related factor CSF1. Behavioral testing before and after treatment in both Alzheimer’s model (5xFAD) and healthy mice showed that the Alzheimer’s model mice displayed the expected movement and anxiety-related changes. They also found differences between males and females in how their brain connections, especially inhibitory synapses, were affected. These findings suggest that IL34 may help shape the brain’s wiring and behavior in sex- and age-specific ways. For Aim 2, the team created a special Alzheimer’s model mouse that enables them to track microglial activity as they gather around amyloid plaques. They have already demonstrated that this tagging system is effective, setting the stage for a deeper analysis of how IL34 might shift microglia from a harmful, overactive state to a healthier, balanced one. Looking ahead, the researchers will finish analyzing excitatory synapses, carry out detailed single-cell studies of microglia, and test whether IL34 or CSF1 can not only alter microglial behavior but also reduce plaques and preserve brain connections. They will also explore whether combining these treatments with anti-amyloid drugs could enhance protection.
