Fig. 1.

Spermidine reduced soluble Aβ and induced transcriptomic alterations in microglia of APPPS1 mice. a APPPS1 mice were treated with 3 mM spermidine via their drinking water starting at 30 days (d) until mice reached an age of 120 days or 290 days according to the depicted treatment scheme. Spermidine-treated APPPS1 mice were compared to non-treated controls (H₂O). The Aβ40 and Aβ42 content was measured in the TBS (soluble) fraction of brain homogenates of 120-day-old or 290-day-old spermidine-treated mice and water controls (mixed sex) using electrochemiluminescence (MesoScale Discovery panel). Values were normalized to water controls. 120d APPPS1 H₂O (n = 14), 120d APPPS1 spermidine (n = 14), 290d APPPS1 H₂O (n = 14), 290d APPPS1 spermidine (n = 12); two‐tailed t‐test, Aβ42 in 120d mice: Mann–Whitney U test. b Single nuclei sequencing of hemispheres harvested from 180-day-old male spermidine-treated APPPS1, H₂O APPPS1 and H₂O control mice was performed of FACS-sorted DAPI-stained nuclei using the 10x Genomics platform (n = 3). c UMAP embedding and clustering of the snRNA-seq data, together with annotation of the major cell types. d Heatmap showing the top 5 marker genes for 300 cells in each of the major cell types. e Dot plot for the top 50 genes in a cell-type-specific differential expression analysis between spermidine-treated APPPS1 and H₂O APPPS1 mice. Color scale indicates log2 fold change, dot size indicates adjusted p value. f Same as e, for selected genes differentially expressed in microglia clusters 1 or 2. Associated pathways are color-coded. g Expression of Plxna2 in APPPS1 spermidine and APPPS1 H₂O mice. Color scale indicates normalized expression, grey dots represent no data (left panels). For validation, neonatal microglia were treated with the indicated concentrations of spermidine in combination with LPS (1 µg/ml) and ATP (2 mM) or with poly I:C (50 µg/ml) and the gene expression was assessed by RT-qPCR (right panels). Plxna2 expression was normalized to Actin and displayed as fold change compared to non-treated control cells; n = 5–6, one-way ANOVA, Dunnett’s post hoc test. h Neonatal microglia were pre-treated with 3 or 10 µM spermidine for 15 h. The confluent cell layer was scratched and the scratch area was imaged for 72 h at the indicated timepoints. The gap area normalized to timepoint 0 h is displayed; n = 5–6, two-way ANOVA, Dunnett’s post hoc test. i Neonatal microglia were non-treated or treated with 10 µM spermidine and their migration towards 300 µM ATP was quantified after 24 h using a transwell migration assay; two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001