Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusionwith mtDNA-less cells

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDN A sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (rho degrees) mouse or hum an cell lines result in viable cybrid cell lines, The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in th e mtDNA COIII, ND3 and tRNA(Arg) genes. The brain origin of the human synap tosome cybrids was confirmed using a rare mtDNA Mbol polymorphism, Fusion o f synaptosomes from the brain of a 35-year-old woman resulted in 71 synapto some cybrids, Sequencing the mtDNA control region of these cybrid clones re vealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs wit h 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable result s were obtained by PCR amplification, cloning and sequencing of mtDNA contr ol regions directly from the patient's brain tissue, but not when the contr ol region was amplified and cloned from a synaptosome cybrid homoplasmic fo r a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control regi on length variants from an adult human brain without recourse to PCR, and e stablished the variant mtDNAs within living cultured cells. This confirms t hat some mtDNA heteroplasmy can exist in human neurons, and provides the op portunity to study its functional significance.