Assessing cellular aging in old and rejuvenated neurons from Alzheimer patients

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Project Summary: Alzheimer’s disease (AD) affects over 50 million people worldwide1. For the vast majority of patients, AD is sporadic, with no known etiology other than advanced age. The field of neuroscience has understandably focused on cell intrinsic properties of neuronal cells as the basis for neurodegenerative diseases, such as AD. However, about half of the cells in the brain are glial cells with ~15% of cells in the brain accounted for by microglia2. Microglia are specialized macrophages of the central nervous system that help to clear debris, pathogens, and neuronal synapses. Under homeostatic conditions, microglia provide support to neurons, while under pathological conditions, microglia often prune synapses excessively, activate astrocytes, increase inflammation, and reduce support to neurons3. Currently, a mechanistic understanding of microglia in Alzheimer’s disease (AD) is unclear. To advance the scientific goals of aim 4, this project will study metabolic neuron-microglia interactions in three-dimensional multicellular cultures. This project will test the hypothesis that aberrant neuronal PKM2 challenges neuron-glia coupling, and that the microglial capacity to prune and respond to inflammatory stimuli is influenced by their APOE (Apolipoprotein E) haplotype. The original proposal indicated that astrocyte activation may be lacking in the absence of microglia to properly activate them. Since, significant advances have been made in the Gage lab to address this issue through the incorporation of microglia into organoids (In press, Cell. May 2023), which will complement the microcarrier data and provide a separate piece of evidence. Metabolic changes in glia by APOE haplotype is well documented4,5, but the impact on neurons is less clear. It is unknown how APOE ε4 allele influences AD onset and progression or how APOE ε2 allele provides protection6. This lab has recently acquired commercially available human isogenic iPSC lines that have been genetically altered to represent each APOE haplotype. Microglia will be generated from each of these lines as previously described7 and cultured in multi-cell microcarriers and organoids. Preliminary data from this lab suggests that microglia with APOE4/4 have AD related metabolic defects associated with lipid metabolism, lysosomal and mitochondrial function. The goal of this project is to analyze the transcriptome and functionality of microglia with different APOE haplotypes. By employing single cell RNA sequencing, this project will identify differences in gene expression on a cell-type basis, between these groups. These data will contribute to the overall proposal and reveal specific genes involved implicated in AD and age- related lysosomal function and metabolism are downregulated by APOE. Through this mechanism, APOE4 microglia may actively contribute to the pathogenesis of AD, rather than solely reacting to AD pathogenesis. The hypothesis of this project is that APOE4 microglia will exhibit decreased mitophagy and lysosomal impairment, therefore increasing toxicity within cells, and ultimately impacting the way APOE4/4 microglia can provide support to neurons, which this project will investigate through the microcarriers and organoid cultures.