Fig. 1
From: Loss of mitochondrial enzyme GPT2 leads to reprogramming of synaptic glutamate metabolism
GPT2 is the primary glutamate pyruvate transaminase in isolated synaptic terminals. A Fractionation of the mouse brain into fractions containing myelin and other membranes, synaptosomes (isolated synaptic terminals) and mitochondria. Note that the synaptosomes are extracted from brain tissue containing only the forebrain, excluding olfactory bulb, midbrain and the brain stem. B Metabolic pathways relevant to GPT2 and glutamate metabolism in synaptosomes including pre-synaptic neuron, post-synaptic neuron and astrocytic components. Ala: alanine, a-keto: alpha-ketoglutarate, Glu: glutamate, Gln: glutamine, Pyr: pyruvate, GDH: glutamate dehydrogenase, GLS: glutaminase, GLUL: glutamine synthetase. C GPT2 is enriched in synaptosomes and mitochondria. Western blotting of the homogenate, cytosolic, myelin and membranes, synaptosomes and mitochondria fractions obtained from a wild-type mouse at postnatal day 18 (P18) for GPT2, GPT1, MTCO1 (mitochondrial marker, subunit 1 of complex IV cytochrome c oxidase), SYN1 (synapsin I), LSD1 (nuclear marker, lysine-specific demethylase 1), GDH (glutamate dehydrogenase), GFAP (glial fibrillary acidic protein) and GAPDH (cytosolic marker, glyceraldehyde 3-phosphate dehydrogenase). D GPT2 is enriched in mitochondria of synaptosomes. Western blotting of synaptosomes that were fractionated further into synaptic membrane, synaptic cytosol and synaptic vesicles obtained from a wild-type mouse at P18 for GPT2, SV2 (synaptic vesicle protein 2), VGLUT1 (vesicular glutamate transporter 1), VGAT (vesicular GABA transporter), GLT1 (glutamate transporter 1, astrocytic), GRIA1 (AMPA receptor subunit 1), ACTIN, GAPDH. E Overall GPT enzyme activity in Gpt2-null is greatly reduced in Gpt2-null synaptosomes at P18. Each dot represents the total enzymatic activities of GPT2 and GPT1 in synaptosome samples obtained from different wild-type (black) or Gpt2-null (red) mice. The enzymatic activities are expressed as mU/mg per protein. P < 0.0001. F Glutamine cannot be used as a precursor for alanine in Gpt2-null synaptosomes at P18. Isotope tracing in wild-type (black) and Gpt2-null (red) synaptosomes at P18 using glutamine labeled with heavy nitrogen at the amine position. Each dot represents fractional enrichment of labeled pool of alanine in synaptosome from a different animal, expressed as percentage of total alanine pool. For pellet, **P = 0.006 and for medium, ***P = 0.00016. G Alanine and alpha-ketoglutarate can be used as precursors for glutamate in wild-type but not in Gpt2-null synaptosomes at P18. Isotope tracing in wild-type (black) and Gpt2-null (red) synaptosomes at P18 using heavy nitrogen labeled alanine. Each dot represents fractional enrichment of labeled pool of glutamate in synaptosome from a different animal, expressed as percentage of total glutamate pool. For pellet and medium, ***P < 0.0001