Combined neocortical protein and morphological profiling of reactive microglia across Alzheimer’s and Creutzfeldt–Jakob disease

Recent RNA-sequencing studies have established a reactive molecular signature and highlighted substantial regional diversity of microglia, underscoring their involvement in neurodegenerative proteinopathies. However, the implications of these findings have not been fully elucidated at the protein expression level in neuropathological settings, especially when comparing different proteinopathies. Using FFPE tissue from postmortem human brains with neuropathologically confirmed sporadic Creutzfeldt-Jakob disease, subtype MM1 (n = 5, formic acid-treated tissue), Alzheimer's disease, Braak stage VI (n = 5), and control brains with no noteworthy pathological changes (n = 2), we (1) verify the reactive microglial signature at the protein expression level utilizing spatial protein profiling, (2) detect a disease-specific amoeboid IBA1+ cell subtype identified with digital morphological profiling, and (3) determine the correlation between identified microglia protein expression profiles and morphology within each and across all brain sample groups. As proof-of-concept, the protein expression and morphology profiling modalities can be bioinformatically integrated to quantify the reactivity of analyzed IBA1+ cells when comparing different neocortical layers (superficial grey matter, deep grey matter, and white matter) and frontal and occipital neocortex across the different diseases. We observed greater microglial reactivity in Creutzfeldt-Jakob disease compared to Alzheimer's disease, and more remarkably, greater reactivity in occipital cortex compared to frontal cortex across both diseases. Both profiling modalities additionally revealed consistent molecular and morphological differences between grey matter and white matter IBA1+ cells, with similar distributional changes observed in the layers across both diseases. This study refines the understanding of canonical, disease-specific, and brain regional features of reactive microglia in two different neurodegenerative proteinopathies and demonstrates the successful application of spatial probe-based protein profiling together with digital morphological profiling on long-term fixed FFPE and even formic acid-treated human brain tissue.