In situ spatial glycomic imaging of mouse and human Alzheimer's disease brains

Abstract

N‐linked protein glycosylation in the brain is an understudied facet of glucose utilization that impacts a myriad of cellular processes including resting membrane potential, axon firing, and synaptic vesicle trafficking. Currently, a spatial map of N‐linked glycans within the normal and Alzheimer's disease (AD) human brain does not exist. A comprehensive analysis of the spatial N‐linked glycome would improve our understanding of brain energy metabolism, linking metabolism to signaling events perturbed during AD progression, and could illuminate new therapeutic strategies. Herein we report an optimized in situ workflow for enzyme‐assisted, matrix‐assisted laser desorption and ionization (MALDI) mass spectrometry imaging (MSI) of brain N‐linked glycans. Using this workflow, we spatially interrogated N‐linked glycan heterogeneity in both mouse and human AD brains and their respective age‐matched controls. We identified robust regional‐specific N‐linked glycan changes associated with AD in mice and humans. These data suggest that N‐linked glycan dysregulation could be an underpinning of AD pathologies.

In Situ Microwave Fixation to Define the Terminal Rodent Brain Metabolome

“…Recently, the glycolysis-lactate axis has been highlighted as a key pathway perturbed in the disease-associated microglia during AD progression 15,16 . Further, we found that protein hyperglycosylation, an anabolic product of glucose metabolism, is a hallmark of AD in both human and mouse specimens 17 . Collectively, these studies highlight the potential of alternative metabolic fates of glucose in neurodegenerative diseases.…”

“…Future studies should focus on isolating single cell types through fluoresce-activated cell sorting (FACS) or magnetic separation methodologies post focused microwave, to define the cell type specific brain metabolism in healthy and diseased tissue. It would also be of interest to test whether focused microwave is compatible with new single-cell technologies such as matrix-assisted laser desorption ionization MALDI mass spectrometry imaging 17 , single cell RNAseq 75 , and spatial proteomics analyses 76,77 .…”

Changes in ProteinN-Glycosylation Regulation Occur in the Human Parkinsonian Brain in a Region-Specific Manner

“…A region-specific modulation of N- glycans upon disease was also seen in this case. For example, FA2G1 (1647 m/z), FA2G2 (1809 m/z) and FA3G1 (1850 m/z) were all shown to be decreased upon AD in the hippocampus, but increased in the cortex from the diseased brains[40]. Interestingly, FMA5A1 (1606 m/z, decreased in the PD striatum) was also decreased in the hippocampus of the AD brains.…”

APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge

“…Slides were used immediately or stored in a desiccator until analysis. For the detection of lipids, a Waters SynaptG2-Xs high-definition mass spectrometer equipped with traveling wave ion mobility was used as previously described 108 . The laser was operating at 2000 Hz with an energy of 300 AU and spot size of 50 μm at X and Y coordinates of 100μm with mass range set at 50 – 1000 m/z in negative mode.…”

“…Brain sections (10 µm) were mounted on glass slides and prepared for MALDI MSI (see Method section "Brain preparation for spatial transcriptomics"). Slides were prepared as previously described 108 . Briefly, after desiccation for one hour, slides were sprayed with 14 passes of 7mg/mL N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDC) matrix (Sigma) in 70% methanol (HPLC-grade, Sigma) was applied at 0.06mL/min with a 3mm offset and a velocity of 1200mm/min at 30°C and 10psi using the M5 Sprayer with a heated tray of 50°C.…”