Furthermore, previously reported genes and novel genes identified in this study and their association with two main AD pathological markers (Aβ plaques and NFTs or neuropil threads) in layers II/III and V are confirmed at the single-cell level by RNAscope HiPlex smFISH assay, including SLC1A3, KIF5A, SNCG, STMN2, CSRP1, PLP1, GLUL, PAQR6, CD9, C1QB, SPP1, CD63, CRYAB, and YWHAH (Fig. 7 and Additional file 1: Fig. S10). Here, CD9 is linked to Alzheimer disease.