The Johnson lab studies the strongest genetic risk factor for Alzheimer’s disease (AD), apolipoprotein E (APOE). The E4 allele of APOE is carried by more than 25% of the population. A single copy of E4 increases Alzheimer’s risk 3-4 fold compared to the more common E3 allele. In homozygous E4 individuals the effect on Alzheimer’s risk is even more dramatic, with a 10-15 fold increase in AD risk compared to E3 homozygotes.

APOE has a long-established role in maintain brain energy homeostasis, which is the focus of work in the Johnson lab. E4 individuals display a distinct pattern of disrupted glucose metabolism in the brain (visible using FDG-PET imaging) as early as their 20s or 30s, decades before disease onset. This same pattern of disrupted glucose metabolism will eventually present in all AD patients and is one of the earliest biomarkers of the disease.

Outside of its central role in metabolism, APOE has also been consistently linked to another prominent feature of Alzheimer’s disease – chronic neuroinflammation. Inflammation in the brain is propagated by ‘glial’ cells such as astrocytes and microglia, and occurs in response to the amyloid plaques and neurodegeneration that characterizes Alzheimer’s pathology. While a healthy inflammatory response is part of the brain’s natural defense system and helps to clear the amyloid plaques, if left unresolved the excessive inflammatory signaling can lead to further damage to nearby neurons. E4 microglia display a pro-inflammatory phenotype with increased secretion of pro-inflammatory cytokines and worsened neuroinflammation in AD.

Nick’s project in the Johnson lab seeks to marry these two facets of APOE’s risk through the concept of ‘immunometabolism’. Immunometabolism is the principle that immune cells such as microglia critically rely on metabolic pathways to perform their immune functions. Pro-inflammatory phenotypes are supported by an increased rate of the glycolysis pathway, whereas anti-inflammatory phenotypes require enhanced mitochondrial respiration. Microglia with defective mitochondria fail to engage an anti-inflammatory response.

Thanks to the tremendous resources available at the UK Sanders-Brown Center on Aging and a network of talented collaborators, the Johnson lab was able to examine this question using state-of-the-art technology. Single cell RNA-sequencing (scRNAseq) enabled the team to probe gene expression in E4 brains on a cell-by-cell basis. This technology offers vast improvements over previous RNA-sequencing tech, where the entire tissue would be ground up and the homogenate run in bulk. Therefore, contributions of individual cell types would often be masked by the overall bulk gene expression. It was impossible to discriminate if the gene expression changes were being driven by neurons, astrocytes, microglia, or any of the myriad other cell types present in the brain. Further, it was not clear where in the brain these genes were being expressed. The Johnson lab combined scRNAseq with another cutting-edge technique called “spatial transcriptomics”, which enables the researcher to visualize precisely in the tissue where each gene is being expressed. Both of these techniques converged on a unique subpopulation of microglia present in the hippocampus and cortex of E4 brains that was highly glycolytic and expressed high levels of several Alzheimer’s risk genes, implicating both immune and metabolic pathways.

In order to validate the disrupted metabolism in E4 microglia, Nick isolated primary microglia from mice and grew them in vitro, where he was able to characterize their metabolic state using two independent methods. In the first, oxygen consumption rates and lactic acid production were measured using “Seahorse extracellular flux analysis”, an assay which enables the researcher to determine metabolic status by comparing mitochondrial respiration to the rate of glycolysis. In the second approach, Nick performed metabolomic analyses on the primary microglia – a technique which uses gas chromatography-mass spectrometry to profile the levels of each metabolite in the cells. Once again, both of these techniques converged on E4 microglia favoring glycolysis for energy production and being deficient in mitochondrial respiration.

Taken together, all four methodologies pointed towards an altered metabolic profile in E4 microglia which supports pro-inflammatory responses, while at the same time preventing anti-inflammatory phenotypes. Such a situation would diminish the capability of the microglia to repair the tissue and would be conducive to chronic neuroinflammation, setting up a vicious cycle whereby the metabolic impairments in E4 microglia propagates inflammation throughout the brains of Alzheimer’s patients and hastens the neurodegeneration. It is Nick’s hope that targeting immunometabolism in E4 microglia by supporting their mitochondrial function holds promise towards resolving neuroinflammation in Alzheimer’s patients, allowing glial cells the opportunity to repair the tissue and slow or halt cognitive decline.

Nick won two awards for his research poster at AAIC 2022. The first award was for a competition hosted by 10x Genomics, the major company supplying single cell RNA-sequencing and spatial transcriptomics technology. Nick won top poster utilizing these technologies at the conference. Nick also won an award for best poster in his “Professional Interest Area” (PIA), a subdivision of the Alzheimer’s Association with a specific focus. Nick won best poster in the Nutrition, Metabolism, and Dementia PIA.