Kim Green, Ph.D. - US grants

DESCRIPTION (provided by applicant): Microglia are implicated in most disorders of the CNS, and while they offer protective roles in aspects of disease, their chronic activation and presence can impede recovery and promote cellular damage and cognitive decline. As such, anti-inflammatory strategies have been pursued as potential therapeutics to many neurodegenerative diseases, but have lacked potency. As part of our investigations into inflammation in the pathogenesis of Alzheimer's disease we targeted the colony-stimulating factor 1 receptor (CSF1R), as this regulates the proliferation of microglia. We discovered that microglia are actually physiologically dependent upon CSF1R signaling, and that administration of CSF1R antagonists results in the rapid and continued elimination of virtually all microglia from the CNS, without gross effects on peripheral macrophage populations. Mice lacking microglia, using this approach, are healthy, viable, and show no deleterious effects (we have treated for up to 3 months thus far). In CNS disease models we find that elimination of microglia is highly beneficial, suggesting that CSF1R antagonists could be an effective therapeutic for most CNS disorders. Crucially, CSF1R antagonists are in clinical trials for various cancers, and thus these findings are translatable. Our first aim is to track the fate of these eliminated microglia - do thy die with CSF1R inhibition, or do they dedifferentiate into a non-microglial cell? We have taken these findings further, to address the fundamental question of the regulation of microglia in the adult brain, by eliminating 99% of microglia via administration of CSF1R antagonists, and then withdrawing the antagonists to see if the microglia population could recover. Astonishingly, IBA-1 positive cells appear throughout the brain after just 3 days, with very different morphologies to resident microglia in control brains. These cells are also positive for CD45, nestin, Ki67 and stain with IB4. None of these markers are present in resident microglia in control brains. By 7 days, these cells have assumed similar morphologies to resident microglia, and these markers are absent once again. Thus, the adult brain has a highly plastic microglia population that can fully replenish itself when depleted, within days. Furthermore, we have identified a non- microglial cell that proliferates throughout the CNS and differentiates into the repopulating microglia - representing a potential microglia progenitor cell. This proposal seeks to further understand the origins of these repopulating cells and further characterize the transition of these potential microglia progenitor cells into microglia. In addition, we will look at the moleculr pathways that signal for repopulation of the microglia- depleted brain, and for differentiation int microglia, once present. Finally, we will assess the therapeutic potential of replacing senescent microglia in the aged brain with repopulating cells, and their ability to improve cognition and clear plaques in mouse models of Alzheimer's disease.

Project 2: Microglia as Mediators of Dendritic Spine Loss and Plaque Formation in the AD Brain Project Summary/Abstract Dendritic spine loss is closely associated with cognitive decline in Alzheimer's disease (AD) and other disorders. Identifying the mechanisms and stimuli that lead to spine loss in disease is crucial to developing strategies to reverse or prevent these losses, hopefully leading to improvements in cognition. Concurrent with spine loss, chronic microglial-activation is found in the AD brain and in other disorders. As part of our investigations into inflammation in the pathogenesis of AD, we targeted the colony-stimulating factor 1 receptor (CSF1R), as this regulates the proliferation of microglia. We discovered that microglia are physiologically dependent upon CSF1R signaling and that administration of CSF1R antagonists results in the rapid and continued elimination of virtually all microglia from the CNS. We have used this approach to determine that microglia do play a highly significant role in regulating dendritic spine numbers in the adult brain elimination of microglia for 8 weeks results in a ~35% increase in spine densities in CA1 and layer V cortical neurons. Additionally, electrophysiology reveals robustly increased excitatory synaptic inputs to neurons, showing direct evidence of increased active synapses. As we have shown that microglia play a role in modulating spine and synapses in the adult brain, we now propose that this normal function goes awry in AD, leading to overpruning of synapses and resulting in reduced spine densities and subsequent cognitive decline. Our project proposes 4 linked aims that will utilize human tissue to explore the relationship between microglia and dendritic spine loss, as well as plaque formation, in AD. Firstly, we will conduct thorough correlations between microglial densities and morphologies with spine loss from post-mortem tissues in control, MCI and AD subjects. We will then test our hypothesis using tissue from high-pathology control subjects. We will utilize human fibroblasts from MCI subjects, with either a high number of AD microglial-risk SNPs or a low number, which are converted to pluripotent stem cells via iPS cell technology, and then differentiated into microglia. We will then explore how microglia derived from these MCI patients differ in their abilities to 1) prune dendritic spines and 2) phagocytose and clear A?, correlating these findings with the conversion into AD from our MCI subjects. Critical to our approach, we now have the technology to eliminate all endogenous microglia from the mouse CNS via administration of CSF1R inhibitors and then repopulate the mouse brain by infusing in human IPS- derived microglia. Using this method, we can explore the effects of these cells in an in vivo setting and thus determine the effects of these human-derived microglia on both dendritic spines and on clearance/formation of A? plaques. Through these experiments we will be able to fully study the relationship between human microglia and AD pathology/spine loss in a fashion that has not been previously possible. These results will potentially lead to the development of inhibitors that can eliminate microglia in the AD brain and hence prevent spine loss.

Recent Genome Wide Association Studies have identified several microglia-expressed genes as conferring increased risk of developing Alzheimer's disease, while activated microglia are a prominent feature of the AD brain. It is critical that we determine how microglia confer risk of development of AD in order to devise appropriate therapeutics. We have developed a method to eliminate all microglia from the adult brain, even for the lifetime of the animal. This elimination allows us to fully study how microglia influence the pathogenesis of AD, and determine their roles in the disease. Our preliminary data has demonstrated that we can completely eliminate microglia from AD mice prior to any pathology development, and we can keep the microglia eliminated for thereafter. We have revealed that microglia are essential for plaque development and in their absence plaques are not formed and instead A? accumulates within the vasculature. Notably, this phenotype resembles that of AD mice containing human ApoE ? the strongest risk factor for AD. This finding places microglia as initiators of the disease and suggests that plaques form because of microglia, and that this is not due to a reduction in microglia-mediated A? clearance. This proposal will fully characterize how pathology develops in the absence of microglia, in multiple mouse models, and determine the mechanisms by which microglia trap A? within the parenchyma and lead to plaque formation. We hypothesize that microglia take up neuronally derived A?, in an ApoE-dependent fashion, and then concentrate it within lysosomal compartments. This buildup leads to microglial cell death, releasing the A?, which then seeds a plaque. Thus, we will explore how microglial cause plaques, and what the role of ApoE is in this process.

Abstract: We discovered microglia in the adult brain are dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R), and identified several CSF1R inhibitors that crossed into the brain, leading to the elimination of most of the microglia. This remarkable phenomenon has been widely replicated and is now a standard in the field to explore microglial function in health and disease, and clinical trials are being conducted/planned as a result. We also found that we could eliminate microglia for as long as we continued treatment, but upon drug withdrawal, repopulation of the microglial tissue occurred rapidly from proliferating cells throughout the brain that formed a new microglial tissue in ~14 days. We found we could use this to ?reset? the inflamed microglial tissue after injury or in aging, and promote functional recovery/cognition. In this continuation, we seek to understand the source and properties of these repopulating cells that become microglia, and study how they modulate neuronal gene expression to rejuvenate the aged brain and fully restore long-term potentiation to that of a young animal. In addition, we describe a second slower source of microglial repopulation, that originates in specific brain niches ? the rostral migratory stream (RMS) and associated projecting axonal tracts. This ?alternative? repopulation is only unmasked by the complete elimination of microglia. These ?alternative? cells arise from unknown cells within these brain niches, and eventually can break out from the white matter tracts and fill the cortex/brain. These cells never attain the numbers, morphologies, or gene expression of microglia, but resemble microglia found in the RMS, which have pro-neurogenesis and increased phagocytotic capabilities than other microglia. We will determine the source of these ?alternative? cells, and the consequences of filling the brain with them, including if they have any therapeutic potential, in a mouse model of Alzheimer's disease.