Extended Data Fig. 3: Related to Fig. 2. Metabolic and structural phenotypes of Letm1 KD neurons.
From: Mitochondrial Ca2+ efflux controls neuronal metabolism and long-term memory across species
(A) Representative images of neurons expressing Syn-ATP. From left to right, neurons are 1) live, 2) permeabilized and with 0 mM ATP and 3) permeabilized and with 5 mM ATP (see methods). (B-C) Non-calibrated values corresponding to main Fig. 2b, shown as ratio of luminescence by fluorescence (L/F). (B) P values were determined using a Kruskal-Wallis test followed by Dunn’s multiple comparisons test (P = 0.018; n = 30 for control, n = 28 for Letm1 KD, n = 7 for Letm1/PDP1 KD. Post hoc P values: Control vs Letm1 KD, **P = 0.0064; Control vs Letm1/PDP1 KD, n.s., P > 0.9999; Letm1 KD vs Letm1/PDP1 KD, *P = 0.0214. (C) ATP levels in the presence of TTX. P value was determined using a two-tailed unpaired two-tailed t-test. (n.s., P > 0.9208; n = 8 for Control + TTX, n = 9 for Letm1 KD + TTX; t15 = 0.1011). Experiments shown in A-C were done in 1.25 mM lactate and 1.25 pyruvate with no glucose. (D) Strategy used to simultaneously KD PDP1 and Letm1. H1 and U6 are RNA Pol III promoters. PGK is a promoter to express mTagBFP2 to confirm transfection. (E) Western blot of pure neuronal cultures to knock-down Letm1 or Letm1 and PDP1 using lentivirus. (F) Quantification of KD for Letm1 and PDP1 using the dual vector strategy. P values were determined using a one-sample Wilcoxon signed rank test (**P = 0.078 for Letm1 KD, *P = 0.0156 for PDP1 KD; n = 8 for Letm1 KD and n = 7 for PDP1 KD; W = -36, -28). (G) Synaptic ATP levels in Control and Letm1 KD neurons upon stimulation with 600APs at 10 Hz. The dotted line indicates baseline ATP levels before stimulation. L/F values are normalized to the baseline. Data are shown as mean±SEM (n = 11). (H) Stimulation induced changes in synaptic ATP levels (ΔL/F) in control and Letm1 KD neurons. P value was determined using a two-tailed unpaired two-tailed t-test (*P = 0.0137; n = 11 for both control and Letm1 KD neurons; t20 = 2.703). (I) Representative Western blot of Letm1 KD pure neurons, showing Letm1, Syphy1 (synaptophysin 1), PSD95, CI-NDUFB8, CII-SDHB, CIII-UQCRC2, COXIV, CV-ATP5A and β-actin. (J) Protein levels are normalized to control. P values were calculated using two-tailed one-sample t-tests: Letm1 (**** P < 0.0001, t₃ = 32.30), Syphy1 (ns P = 0.5755, t₃ = 0.6263), PSD95 (ns P = 0.0659, t₃ = 2.835), Complex I (ns P = 0.6821, t₃ = 0.4518), Complex II (n.s., P = 0.5133, t₃ = 0.7394), Complex III (n.s., P = 0.7529, t₃ = 0.3450), Complex IV (n.s., P = 0.2027, t₃ = 1.625), Complex V (n.s., P = 0.4177, t₃ = 0.9375). (K-M) Axonal mitochondrial shape properties measured using Mito4x-GCaMP6f. Circularity is measured in arbitrary units (a.u.), 1 = circular; 0 = non-circular. P values were determined using two-tailed Mann–Whitney U-tests for circularity (n.s., P = 0.0925; n = 29 neurons for control, n = 22 for Letm1 KD; U = 230) and size (*P = 0.0160; n = 29 neurons for control, n = 22 for Letm1 KD; U = 193), and using a two-tailed unpaired t-test for mitochondria number (*P = 0.0115, n = 8 for both conditions; t14 = 2.906). (N) Number of synapses per 100 µm measured from synaptophysin-mCherry signal from SynATP. P values were determined using a ordinary one-way ANOVA (**P = 0.0019; F(2,24) = 0.2812; n = 9 for control, n = 11 for Letm1 KD, n = 7 for Letm1/PDP1 KD, followed by Dunn’s multiple comparisons test. Post hoc P values: Control vs Letm1 KD, *P = 0.0135; Control vs Letm1/PDP1 KD, n.s., P = 0.7126; Letm1 KD vs Letm1/PDP1 KD, **P = 0.0032). (O) Estimated synapse size, shown as area in µm2. P values were determined using a one-way ANOVA with n = 23 for control, n = 22 for Letm1 KD, n = 7 for Letm1/PDP1 KD, followed by Tukey’s multiple comparisons test. Post hoc P values: Control vs Letm1 KD, **P = 0.0031; Control vs Letm1/PDP1 KD, n.s., P > 0.9999; Letm1 KD vs Letm1/PDP1 KD, *P = 0.0469. (P) Quantification of synaptic vesicle cycling using vGlut-pHluorin. The figure shows traces corresponding to the mean ± SEM of responses normalized to the total synaptic vesicle pool, obtained by applying NH4Cl at pH 7.4. P value was determined using a two-tailed Mann Whitney U-test (n.s. P = 0.6126; n = 8 for control neurons and n = 7 Letm1 KD; U = 23). (Q) Comparison of synaptic vesicle pH estimates using vGlut-pH. P value was determined using a two-tailed unpaired two-tailed t-test (n.s. P = 0.66; n = 8 for both control and Letm1 KD neurons; t14 = 0.4389).
