Periodic Reporting for period 1 - MicroSyn-MIND (Microglia-synapse molecular interactions in neurodegenerative disorders)
Período documentado: 2022-08-01 hasta 2024-07-31
Alzheimer’s disease (AD) affects over 43.8 million people worldwide, leading to the progressive loss of neurons and their connections (synapses). This results in memory loss, cognitive decline, and disconnection from reality, imposing a significant burden on patients and society. While various factors contribute to AD progression—ranging from genetic risks to the accumulation of extracellular Amyloid-β (Aβ) plaques, tau tangles within neurons, and changes in glial cells and blood vessels—the precise mechanisms remain unclear. For instance, Aβ plaques can accumulate years before symptoms appear and do not always lead to neurodegeneration. We hypothesize that microglia, the brain’s immune cells, play a crucial role in AD. Microglia protect and support neurons but can also prune excess synapses during early brain development. In AD, we suspect that microglia not only target Aβ plaques but also healthy synapses, contributing to neuronal loss. However, the interactions between microglia, neurons, and synapses, and how these interactions change in AD, are not well understood. Previous studies suggest that specific receptors and ligands on microglia and synapses mediate these interactions, making them potential drug targets. While some of these proteins are known to regulate synaptic pruning, others remain undiscovered. Most knowledge about microglia comes from rodent studies, but human microglia behave differently, particularly in expressing AD risk genes and responding to Aβ. To address this, we transplanted human stem cell-derived microglia into the brains of mice (xenotransplantation), allowing human microglia to develop in a living brain environment and adopt human-like behaviors, especially in response to Aβ plaques. Our main objective is to use this xenotransplantation model to identify human proteins involved in microglia-synapse interactions and determine if changes in these proteins lead to synapse loss in AD. We employ techniques like TurboID and mass spectrometry to tag and identify these proteins, creating an interactome map. This map will be a valuable resource for the microglia research field. Next, we aim to investigate how Aβ plaques affect these interactions and specifically how they influence synapse loss. This research could uncover new therapeutic targets to prevent synapse loss in AD, significantly impacting treatment strategies and improving the quality of life for AD patients. The expected impacts of this project are substantial. By elucidating the role of microglia in AD and identifying new therapeutic targets, we aim to advance treatment strategies significantly. This could lead to improved outcomes and quality of life for millions of AD patients worldwide, highlighting the project’s scale and significance.
Our project aimed to elucidate how Alzheimer’s disease (AD) affects the interactions between microglia and synapses. We are currently working on creating a detailed map of these interactions in both healthy and AD-affected brains. This task has proven more challenging than anticipated due to several factors. Initially, we planned to use a published technique that expresses TurboID on the cellular outer membrane. However, this approach proved incompatible with our model system. We experimented with various delivery methods, including plasmids, viral vectors, and promoters, alongside multiple rounds of microglial xenotransplantation. These efforts led us to conclude that the original extracellular TurboID technique was not viable. We then shifted to a two-part alternative approach:
1. Inner-membrane turboID: This method aims to identify membrane-bound proteins of microglia in living systems.
2. Data analysis: We analyzed previously generated datasets to identify potential receptors and ligands on microglia and neurons.
Additionally, we are transplanting healthy and AD-model mice with healthy human microglia to isolate and analyze microglial membrane fractions after AD-like symptoms appear. This alternative method aims to create an interactome map and identify potential therapeutic targets. Despite the challenges, we have made significant progress:
• Novel construct designs: We optimized new constructs for both in vitro and in vivo experiments.
• Stable-expression iPSCs: We confirmed that using stable-expression induced pluripotent stem cells (iPSCs) enhances efficiency.
• Initial proteomics datasets: We generated initial proteomics datasets, contributing to a comprehensive atlas of microglial compartment-specific proteomes.
We continue to identify potential microglial cell surface proteins that could be targeted to study their role in synaptic phagocytosis (the process by which cells engulf and remove synapses). Using our revised two-part strategy, we are confident in our ability to identify and knock out specific microglial proteins to assess their effects on synaptic phagocytosis. Although we have encountered challenges, our flexible approach and optimization of techniques will help us achieve our objectives and contribute valuable insights into the mechanisms of synaptic loss in AD in the near future.
1. Inner-membrane turboID: This method aims to identify membrane-bound proteins of microglia in living systems.
2. Data analysis: We analyzed previously generated datasets to identify potential receptors and ligands on microglia and neurons.
Additionally, we are transplanting healthy and AD-model mice with healthy human microglia to isolate and analyze microglial membrane fractions after AD-like symptoms appear. This alternative method aims to create an interactome map and identify potential therapeutic targets. Despite the challenges, we have made significant progress:
• Novel construct designs: We optimized new constructs for both in vitro and in vivo experiments.
• Stable-expression iPSCs: We confirmed that using stable-expression induced pluripotent stem cells (iPSCs) enhances efficiency.
• Initial proteomics datasets: We generated initial proteomics datasets, contributing to a comprehensive atlas of microglial compartment-specific proteomes.
We continue to identify potential microglial cell surface proteins that could be targeted to study their role in synaptic phagocytosis (the process by which cells engulf and remove synapses). Using our revised two-part strategy, we are confident in our ability to identify and knock out specific microglial proteins to assess their effects on synaptic phagocytosis. Although we have encountered challenges, our flexible approach and optimization of techniques will help us achieve our objectives and contribute valuable insights into the mechanisms of synaptic loss in AD in the near future.
Our project has made significant progress, providing new insights into the study of human microglia in both healthy and AD-affected brains. Although we have not yet fully mapped the microglia-synapse cell surface proteins as originally proposed, we have secured additional time and resources to complete this work. We are confident that we will deliver the expected results and impacts in the near future. While the final results, including the interactome map and new potential treatment targets, are still forthcoming, we have achieved several notable advancements:
• New plasmid designs: We have developed various new plasmid designs that enable TurboID expression in different cellular sub-compartments. These designs are applicable to any cell type derived from human stem cells.
• Stable iPSC lines: We have generated a wide range of induced pluripotent stem cell (iPSC) lines that stably express these TurboID constructs under antibiotic selection. These lines can be differentiated into any cell type of interest and will be made available to the research community in the near future.
• Model limitations: We have identified clear limitations in applying previously published techniques to advanced models, such as our MIGRATE xenotransplantation model. This finding is valuable to the field as it highlights the need for further refinement of these techniques.
With the additional time and resources secured for the primary investigator, we are on track to complete the project as originally proposed. The outcomes of this project are expected to significantly advance our understanding of microglia-synapse interactions and identify new therapeutic targets for AD, ultimately contributing to improved treatment strategies and patient outcomes.
• New plasmid designs: We have developed various new plasmid designs that enable TurboID expression in different cellular sub-compartments. These designs are applicable to any cell type derived from human stem cells.
• Stable iPSC lines: We have generated a wide range of induced pluripotent stem cell (iPSC) lines that stably express these TurboID constructs under antibiotic selection. These lines can be differentiated into any cell type of interest and will be made available to the research community in the near future.
• Model limitations: We have identified clear limitations in applying previously published techniques to advanced models, such as our MIGRATE xenotransplantation model. This finding is valuable to the field as it highlights the need for further refinement of these techniques.
With the additional time and resources secured for the primary investigator, we are on track to complete the project as originally proposed. The outcomes of this project are expected to significantly advance our understanding of microglia-synapse interactions and identify new therapeutic targets for AD, ultimately contributing to improved treatment strategies and patient outcomes.