Journal: Cell reports

Article Title: Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells

doi: 10.1016/j.celrep.2018.09.027

Figure Lengend Snippet:

Article Snippet: FLHKI-pGFPN3 and FLHKII-pGFPN3 were a gift from Hossein Ardehali (Addgene plasmid # 21917 and # 21920) ( ). pcDNA3.1 FLII 12 Pglu-700μδ6 was a gift from Wolf Frommer (Addgene plasmid # 17866) ( ).

Techniques: Recombinant, Transfection, XF Assay, Reverse Transcription, SYBR Green Assay, Software

Journal: bioRxiv

Article Title: “A feed-forward Ca 2+ -dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

doi: 10.1101/2021.02.02.429391

Figure Lengend Snippet: A: 25 μM NMDA-stimulated respiration in cortical neurons incubated in DMEM and 2.5 mM glucose, in the presence of 2 mM Ca 2+ (+Ca 2+ ) or without calcium (-Ca 2+ ; plus 100 μM EGTA), expressed as percentage of basal values (OCR %). Mitochondrial function was determined through sequential addition of metabolic inhibitors: 6 μM Olig, 0.5 mM DNP and 1 μM/1 μM A/R at the indicated time points. B: Quantification of NMDA-stimulated OCR (%); mean ± SEM (bars), n = 9-16 (dots) from two different experiments; one-way ANOVA, *** p ≤ 0.001, post hoc Bonferroni test. C: Changes in mitochondrial Ca 2+ (Ca 2+ -mit) in Scrambled ( Scr ) and Mcu -silenced ( Mcu -KD) neurons transfected with 4mtD3cpv probe, after 25 μM NMDA stimulation. D: Quantification of maximum Ca 2+ -mit increment (ΔF max) after NMDA stimulation. Data are mean ± SEM (bars), n = 10-11 (dots) from three independent experiments; T-test *** p -value = 0.0006 E: 25 μM NMDA-stimulated respiration in Scrambled and Mcu -KD neurons, expressed as percentage of basal values (OCR %); F: Quantification of NMDA-stimulated OCR (%); mean ± SEM (bars), n = 30-33 (dots) from 5 independent experiments; T-test * p -value = 0.0173. G: 25 μM NMDA-stimulated respiration in wild type and Aralar -KO neurons, expressed as percentage of basal values (OCR %); H: Quantification of NMDA-stimulated OCR (%); mean ± SEM (bars), n = 9-16 (dots) from 2 independent experiments; T-test **** p -value < 0.0001. I, J: Neurons transfected with Pyronic (I) and Laconic (J) probes to determine changes in cytosolic pyruvate (Pyr) and lactate (Lac), respectively, upon (+Ca 2+ ) or absence (-Ca 2+ ) of calcium. K, L: Dose-dependent effect of 5 μM and 25 μM NMDA induction of Pyr (K) and Lac (L) production. Data are mean ± SEM, n = 5-15 neurons per condition from 2-6 independent experiments. M. Intracellular pH variation determined in BCECF-AM-loaded neurons after 5 mM lactate (Lac) acute addition with or without 1 min pre-incubation with 1 μM MCT1/2 inhibitor AR-C155858 (AR-C1). Data are mean ± SEM, n = 6 per condition from 2 independent experiments. N: Lactate changes induced by 25 μM NMDA in neurons with a pre-addition of HCSS (Veh) of 1 μM AR-C1. O: Quantification of maximum fluorescence change (ΔF max) in Lac after Veh/1 μM AR-C1 and 25 μM NMDA. P: Lactate changes induced by 25 μM NMDA in neurons with extracellular 2 mM lactate (Lact) or in control (Ctr); Q: Pyruvate changes induced by 25 μM NMDA in neurons pre-incubated 30 min with HCSS (Veh), 0.5 mM iodoacetate (IAA) or IAA + 1 μM AR-C1 (IAA + AR-C1). Data are mean ± SEM, n = 8-20 per condition from 4-8 independent experiments for Pyronic and Laconic assays; 5 mM Pyr or Lac was added 3 min after NMDA as a control. The 4mtD3cpv, Pyronic, Laconic and BCECF-AM experiments were performed in HCSS, 2.5 mM glucose and 2 mM Ca 2+ or 100 μM EGTA (-Ca 2+ ).

Article Snippet: Primary cortical neurons, seeded on chambered coverglass 4-well plates (Lab-Tek) at 1.5 × 10 5 cells/well density, were transfected at DIV 7-8 using calcium phosphate protocol with 1 µg of plasmids coding fluorescent sensors for the measurement of cytosolic glucose [FLII 12 Pglu-700μδ6; Addgene #28002 ( )], pyruvate [Pyronic; Addgene #51308 (San Martin et al., 2014)], lactate [Laconic; Addgene #44238 (San Martin et al., 2013)], and for mitochondrial Ca 2+ determinations [4mt-D3cpv; Addgene #58184 ( )].

Techniques: Incubation, Transfection, Fluorescence

Journal: bioRxiv

Article Title: “A feed-forward Ca 2+ -dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

doi: 10.1101/2021.02.02.429391

Figure Lengend Snippet: A-D: Changes in cytosolic Ca 2+ (Ca 2+ -cyt) after 5 (A, C) and 25 μM NMDA (B, D) stimulation in Scr (A, B) or Mcu-KD (C, D) neurons. E-I: Changes in cytosolic Ca 2+ (Ca 2+ -cyt), after 5 (E, G) and 25 μM (F, H) NMDA in wild type (E, I) and Aralar -KO (G, H) Fura2-AM loaded neurons. Individual cell recordings (grey) and average (black) are shown. I: Quantification of peak amplitude as ΔRatio (F 340 /F 380 ) ± SEM (bars) comparing 5 and 25 μM NMDA stimulus; two-way ANOVA, ** p ≤ 0.01,*** p ≤ 0.001, post hoc Bonferroni test. Recordings from at least 50 cells/condition from 4-7 wells (dots) and two independent experiments. J-L: Changes in mitochondrial Ca 2+ (Ca 2+ -mit), in wild type (J) and Aralar -KO neurons (K) transfected with 4mtD3cpv probe, after 5 and 25 μM NMDA stimulation; L: maximum Ca 2+ -mit increment (ΔF max) after NMDA stimulation. Mean ± SEM (bars), 5-10 cells (dots) from two independent culture; two-way ANOVA, * p ≤ 0.05, post hoc Bonferroni test. All cells were maintained in 2.5 mM glucose and 2 mM Ca 2+ HCSS.

Article Snippet: Primary cortical neurons, seeded on chambered coverglass 4-well plates (Lab-Tek) at 1.5 × 10 5 cells/well density, were transfected at DIV 7-8 using calcium phosphate protocol with 1 µg of plasmids coding fluorescent sensors for the measurement of cytosolic glucose [FLII 12 Pglu-700μδ6; Addgene #28002 ( )], pyruvate [Pyronic; Addgene #51308 (San Martin et al., 2014)], lactate [Laconic; Addgene #44238 (San Martin et al., 2013)], and for mitochondrial Ca 2+ determinations [4mt-D3cpv; Addgene #58184 ( )].

Techniques: Transfection

Journal: bioRxiv

Article Title: “A feed-forward Ca 2+ -dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

doi: 10.1101/2021.02.02.429391

Figure Lengend Snippet: A, D: Cortical neurons from wild type and Aralar -KO mice transfected with Pyronic (A) and Laconic (D) probes were stimulated with 25 μM NMDA in 2.5 mM glucose HCSS, in the presence of 2 mM Ca 2+ (+Ca 2+ ) or 100 μM EGTA (-Ca 2+ ). FRET changes reporting cytosolic pyruvate or lactate levels are shown. B, E: Quantification of maximum fluorescence change (ΔF max) in Pyr (B) and Lac (E) after NMDA stimulation. C, F: Quantification of velocity of increase of Pyr (C) and Lac (F) as the increment of fluorescence ratio during the first 30 seconds after stimulation (ΔF/min). n = 10-22 per condition from 5-8 independent experiments for all Pyr and Lac assays; 5 mM Pyr or Lac was added 3 min after NMDA as a control. Data are mean ± SEM, T-test * p = 0.0133.

Article Snippet: Primary cortical neurons, seeded on chambered coverglass 4-well plates (Lab-Tek) at 1.5 × 10 5 cells/well density, were transfected at DIV 7-8 using calcium phosphate protocol with 1 µg of plasmids coding fluorescent sensors for the measurement of cytosolic glucose [FLII 12 Pglu-700μδ6; Addgene #28002 ( )], pyruvate [Pyronic; Addgene #51308 (San Martin et al., 2014)], lactate [Laconic; Addgene #44238 (San Martin et al., 2013)], and for mitochondrial Ca 2+ determinations [4mt-D3cpv; Addgene #58184 ( )].

Techniques: Transfection, Fluorescence

Journal: bioRxiv

Article Title: “A feed-forward Ca 2+ -dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

doi: 10.1101/2021.02.02.429391

Figure Lengend Snippet: Cytosolic pyruvate (Pyr) (A, C) and lactate (Lac) (B, D) levels of Scr and Mcu -KD silenced neurons transfected with Pyronic and Laconic probes, respectively, upon neuronal stimulation with 5 μM (A, B) or 25 μM NMDA (C, D). Neurons were incubated in 2.5 mM glucose and 2 mM Ca 2+ HCSS. Data are mean ± SEM, n = 7-15 neurons per condition from 2 independent cultures.

Article Snippet: Primary cortical neurons, seeded on chambered coverglass 4-well plates (Lab-Tek) at 1.5 × 10 5 cells/well density, were transfected at DIV 7-8 using calcium phosphate protocol with 1 µg of plasmids coding fluorescent sensors for the measurement of cytosolic glucose [FLII 12 Pglu-700μδ6; Addgene #28002 ( )], pyruvate [Pyronic; Addgene #51308 (San Martin et al., 2014)], lactate [Laconic; Addgene #44238 (San Martin et al., 2013)], and for mitochondrial Ca 2+ determinations [4mt-D3cpv; Addgene #58184 ( )].

Techniques: Transfection, Incubation

Journal: bioRxiv

Article Title: “A feed-forward Ca 2+ -dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”

doi: 10.1101/2021.02.02.429391

Figure Lengend Snippet: A: Changes in cytosolic pH in BCECF-loaded neurons after 25 μM NMDA and 5 mM HCl acute addition. B: Changes in glucose levels in neurons transfected with FLII 12 Pglu-700μδ6 probe, after 25 μM NMDA and 5 mM HCl acute addition. Data are mean ± SEM, recordings from 5-6 cells per condition. C, F: 25 μM NMDA induced changes in glucose levels in wild type (C) and Aralar -KO (F) neurons, with or without calcium or with a 20 seconds pre-incubation with 50 μM cytochalasin B (CytB) or 0.5 mM iodoacetate (IAA). D, G: Velocity of increase of glucose levels during the first 30 seconds after stimulation (ΔF/min) in wild type (D) and Aralar -KO (G) neurons. E, H: FRET ratio change between pre-addition of NMDA and the end of the recording (ΔF end point) in wild type (G) and Aralar -KO (H) neurons. Mean ± SEM; recordings from 7-17 cells per condition from 3-5 independent experiments. One-way ANOVA, * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001, post hoc Bonferroni test. ΔF at the end point after NMDA addition is higher in Aralar -KO than in wild type neurons; two-way ANOVA, ** p ≤ 0.005, post hoc Bonferroni test. Assays using BCECF and FLII 12 Pglu-700μδ6 probes were performed in 2.5 mM glucose and 2 mM Ca 2+ (+Ca 2+ ) or 100 μM EGTA (-Ca 2+ ) HCSS.

Article Snippet: Primary cortical neurons, seeded on chambered coverglass 4-well plates (Lab-Tek) at 1.5 × 10 5 cells/well density, were transfected at DIV 7-8 using calcium phosphate protocol with 1 µg of plasmids coding fluorescent sensors for the measurement of cytosolic glucose [FLII 12 Pglu-700μδ6; Addgene #28002 ( )], pyruvate [Pyronic; Addgene #51308 (San Martin et al., 2014)], lactate [Laconic; Addgene #44238 (San Martin et al., 2013)], and for mitochondrial Ca 2+ determinations [4mt-D3cpv; Addgene #58184 ( )].

Techniques: Transfection, Incubation