Journal: Science Advances

Article Title: Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission

doi: 10.1126/sciadv.adp7423

Figure Lengend Snippet: ( A and C ) Co-immunostaining of hippocampal neurons with antibodies against MPC1 (A) or MPC2 (C) and the presynaptic protein vGLUT1. Scale bars, 10 μm. ( B and D ) Magnification of the boxed areas in (A) and (C). Colocalization denoted with arrowheads. Scale bars, 10 μm (B) and 5 μm (D). ( E ) Representative images of axonal mitochondria expressing a mitochondrial pyruvate sensor, CamKII-mtPyronicSF, showing an increase in fluorescence intensity with perfusion of 10 mM extracellular pyruvate compared to 0 mM. Scale bar, 10 μm. ( F ) Average traces of CamKII-mtPyronicSF (Δ F / F 0 ) showing inhibition of mitochondrial pyruvate uptake in neurons expressing shRNA against mpc1 (MPC1 KD). F: fluorescence intensity. n = 12 to 18 (neurons). ( G ) Maximal CamKII-mtPyronicSF fluorescence response in 10 mM pyruvate as determined from traces in (F). ( H ) Schematic of the pyruvate sensor CaMKII-mtPyronicSF and the ATP indicator Syn-ATP expressed in nerve terminals. ( I ) Presynaptic ATP traces in terminals supplied with lactate and pyruvate (lac/pyr) after incubation with the MPC inhibitor UK5099 (100 μM), normalized to preincubation levels. n = 9 (neurons). ( J ) Presynaptic ATP level after application of MPC inhibitor UK5099 normalized to ATP level before. The box-whisker plots denote median (line), 25th to 75th percentile (box), and minimum-maximum (whiskers). Man-Whitney U test (G) and one sample t test (J). Error bars are SEM.

Article Snippet: Neurons were then permeabilized with 0.2% Triton X-100, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature (RT), and incubated at RT for 2 hours or overnight at 4°C with the following primary antibodies against: MPC1 (1:500; rabbit, Sigma-Aldrich, HPA045119), MPC2 (1:500; rabbit, Thermo Fisher Scientific, 20049-1-AP), and vGLUT1 (1:500; guinea pig; Sigma-Aldrich, AB5905).

Techniques: Immunostaining, Expressing, Fluorescence, Inhibition, shRNA, Incubation, Whisker Assay

Journal: Science Advances

Article Title: Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission

doi: 10.1126/sciadv.adp7423

Figure Lengend Snippet: Key resource table. ATCC, American Type Culture Collection; IgG, immunoglobulin G; N/A, not applicable.

Article Snippet: Neurons were then permeabilized with 0.2% Triton X-100, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature (RT), and incubated at RT for 2 hours or overnight at 4°C with the following primary antibodies against: MPC1 (1:500; rabbit, Sigma-Aldrich, HPA045119), MPC2 (1:500; rabbit, Thermo Fisher Scientific, 20049-1-AP), and vGLUT1 (1:500; guinea pig; Sigma-Aldrich, AB5905).

Techniques: Western Blot, shRNA, Inhibition, Magnetic Beads, Plasmid Preparation, Sequencing, CRISPR, Software