Figure 4

Changes in synaptic and mitochondrial protein levels during torpor and arousal. (A) After torpor induction, hippocampal protein samples were prepared from EU, TL and AL mice (n = 6 per group). P2 fractions were used for quantitative mass spectrometry. Significantly regulated proteins were validated with Western blotting, and ontology enrichment was used for functional interpretation. (B) In total, 3764 proteins were quantified. Focusing on three contrasts, TL versus EU, AL versus EU and AL versus TL, 138 (54 up and 84 down), 83 (32 up and 51 down) and 194 (133 up and 61 down) significantly regulated proteins were identified after multiple testing correction (FDR; q < 0.05). (C) Volcano plots showing the -10log p-value (y-axis) and the log2 fold change (x-axis) of all quantified proteins in the three different contrasts. Dashed lines indicate the p < 0.05 cutoff (Student’s t-test), FDR corrected significant proteins with q < 0.05 are depicted as filled dots. Colors indicate proteins that were annotated to the synapse (green), mitochondrion (orange), both (blue) or other cellular components (black). (D) Downregulated proteins in TL versus EU were typically upregulated in AL versus TL (left), and upregulated proteins in AL versus TL were typically also upregulated in AL versus EU (right). Dashed lines depict mean regulation. The red box marks ‘AL overshoot’ proteins (n = 99) that are upregulated from TL to AL, and in AL show significantly higher expression than in EU controls. (E) Pie charts showing a significant overrepresentation of the cellular component term synapse (green) in ‘AL overshoot’ proteins compared with all quantified proteins (Fisher’s Exact test; p < 0.001). Mitochondrion (orange) is the second most abundant term in this group, although not significantly enriched. (F-M) Functional annotation and enrichment of synaptic proteins were further determined with SynGO. Postsynaptic proteins in particular show relatively high counts in downregulated proteins in TL versus EU (F), in upregulated proteins in AL versus TL (G) and AL versus EU (H), and in ‘AL overshoot’ proteins (I). (J-M) Significant enrichment of synaptic SynGO terms was observed primarily in upregulated protein groups (AL vs TL, AL vs EU and ‘AL overshoot’; q < 0.05), and were predominantly postsynaptic in nature and associated with the postsynaptic density (PSD).