Fig. 5

Luteolin increases mitochondrial Ca²⁺ levels, Ca²⁺ transfer from ER, and bioenergetics in synaptosomes in an IP₃R-dependent manner. a Representative traces of mitochondrial (mt-Ca²⁺, upper panel) and cytosolic (cyt-Ca²⁺, lower panel) Ca²⁺ levels in cortical neurons. The arrow indicates the addition of a mix of IP₃-generating agonists (100 μM ATP, 300 μM carbachol, and 100 μM glutamate) in a Ca²⁺-free, EGTA-containing solution. b–d Basal mt-Ca²⁺ levels, mean peak area of mt-Ca²⁺ and cyt-Ca²⁺ were quantified in cortical neurons treated with DMSO or luteolin (2.5 μM, 16 h) (in b, n = 121 Ctr, n = 147 Lut 2.5 μM; in c, n = 33 Ctr, n = 28 Lut 2.5 μM; in d, n = 40 Ctr, n = 43 Lut 2.5 μM from 3 independent cultures). e, f Schematic representation of PDH activation by calcium (in e). In the presence of calcium, PDH phosphatases (PDPs) are activated, favoring PDH dephosphorylation and activation. Phosphorylated and total levels of PDH E1α were quantified in neurons treated with DMSO or luteolin (2.5 μM, 16 h) by western blotting using specific antibodies (n = 5–6; each point in the graph represents one independent experiment). g NADH levels were quantified in mitochondrial-enriched extracts obtained from cortical neurons treated with DMSO or luteolin (2.5 μM, 16 h) (n = 6 run in triplicates; each point in the graph represents one independent experiment). h, i Electron flow was measured in cortical mitochondria isolated from WT mice using a Seahorse flux analyzer after a short 30 min incubation with luteolin or DMSO. Mitochondrial complex inhibitors or substrates, 2 μM Rot, 10 mM succinate (Succ), 4 μM Ant A, and 1 mM ascorbate (Asc)/ 100 mM TMPD, were sequentially injected to calculate mitochondrial complex I–IV activities, respectively (n = 4; each point in the graph represents data from one animal’s brain). j, k Pure synaptosome preparations (represented in j) were incubated for 30 min with luteolin (2.5 μM) and/or XeC (5 μM), and total ATP levels were quantified by luminescence. Ant A (3.2 μM, 30 min) was used as a positive control, which decreased ATP levels by 56% (n = 5; each point in the graph represents data from one animal’s brain). Scale bar = 1 μm (upper image); 200 nm (inset). Statistical significance: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 by Mann-Whitney test when comparing two independent samples (in b, c, f, i). ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 by non-parametric Kruskal-Wallis test (in k)