Characterization of glial responses in Alzheimer’s disease with cyclic multiplex fluorescent immunohistochemistry and machine learning

Background Aβ plaques and neurofibrillary tangles in Alzheimer’s disease (AD) are associated with reactive astrocytes and microglia. Recent single nuclei RNA‐seq studies have reported several transcriptomic phenotypes or states of these glial cells in normal aging and AD. We tested whether multiple astrocyte and microglia states can be detected in human postmortem brain sections from cognitively healthy control and AD subjects using a novel cyclic multiplex fluorescent immunohistochemistry protocol. Method Single formalin‐fixed paraffin‐embedded sections from the temporal association cortex (BA38) of n=7 AD and n=7 control individuals were subjected to 8 cycles of multiplex fluorescent immunohistochemistry, totaling 16 primary antibodies (astrocytes: ALDH1L1, EAAT1/GLAST, EAAT2/GLT‐1, GFAP, glutamine synthetase [GS], TSPO, vimentin, and YKL‐40; microglia: CD68, ferritin, IBA1, MHC2, TMEM119, and TSPO; neurons: HuC/D; and Aβ/phospho‐tau). Between successive cycles, we scanned sections in an epifluorescence microscope, denatured/stripped antibodies, and quenched remaining fluorescence. Our analysis pipeline consisted of: (1) image alignment and co‐registration across cycles; (2) background subtraction; (3) manual annotation of individual ALDH1L1+ astrocytes and IBA1+ microglia; (4) local thresholding and segmentation, resulting in 5,172 astrocytic and 6,226 microglial profiles; and (5) unsupervised spectral clustering on the normalized intensity values for the above combinations of markers. Result We identified three broad phenotypes of astrocytes and microglia: “homeostatic,” “intermediate,” and “reactive.” Homeostatic and reactive glia were predominant in control and AD subjects, respectively, whereas intermediate were equally distributed across both groups. Compared to homeostatic, reactive astrocytes contain substantially higher GFAP and YKL‐40 levels, modestly elevated vimentin and TSPO as well as EAAT1/GLAST, and reduced GS. Intermediate astrocytes are defined by markedly increased EAAT2/GLT‐1, moderately increased GS, and intermediate GFAP and YKL‐40 levels. Relative to homeostatic, reactive microglia show increased expression of all markers, including surprisingly TMEM119, whereas intermediate microglia exhibit increased ferritin and TMEM119 as well as intermediate CD68 levels. Conclusion Cyclic multiplex fluorescent immunohistochemistry allows the deep phenotyping of glia at single‐cell resolution in postmortem brain specimens to identify distinct s