IBIMOL   23987
INSTITUTO DE BIOQUIMICA Y MEDICINA MOLECULAR PROFESOR ALBERTO BOVERIS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Isolation of brain synaptosomes with functional mitochondria
Autor/es:
ANALIA G. KARADAYIAN; SILVIA LORES ARNAIZ; JUAN IGNACIO GUERRA; PAULINA LOMBARDI; JUANITA BUSTAMANTE; ANALIA G. KARADAYIAN; SILVIA LORES ARNAIZ; JUAN IGNACIO GUERRA; PAULINA LOMBARDI; JUANITA BUSTAMANTE
Lugar:
Villa Carlos Paz, Córdoba
Reunión:
Congreso; XXXIV Reunión Anual SAN 2019; 2019
Institución organizadora:
Sociedad Argentina de Investigación en Neurociencias
Resumen:
Mitochondrial function in nerve terminals is essential to maintain adequate neuronal function. The analysis of bioenergetics at synapses is relevant for the study of mitochondrial alterations in neuronal diseases. Hence, we propose a method for brain synaptosomes isolation with functional mitochondria. Mouse brain cortexes from two mice were homogenized and centrifuged at 1000 g for 10 min. The supernatant was layered on a gradient of 3, 8 and 13% Ficoll and centrifuged at 40000 g for 11 min. Mitochondrial function and synaptosomes structural and functional properties were analyzed in the synaptosomal fraction. The presence of intact synaptosomes was detected by flow cytometry after immunostaining with SNAP-25/FITC. Enzymatic activity of acetylcholinesterase and mitochondrial complexes I-III and II-III was measured both in synaptosomal fractions and in total homogenates. Data showed a 1.2-1.4-fold enrichment of acetylcholinesterase and mitochondrial respiratory complexes enzymes relative to the initial homogenate. Oxygen consumption was determined in synaptosomes showing a basal respiration of 11 ± 1 ng at O/min.mg protein, which responded to the addition of oligomycin (5.1 ± 0.4) and FCCP (14.9 ± 0.5). The use of TMRE by flow cytometry indicated a detectable mitochondrial membrane potential which decreased 90% after addition of FCCP. We conclude that the present isolation protocol constitutes an easy and fast method to obtain synaptosomes containing functional mitochondria.