LETM1 is located in the chromosomal region that is deleted in patients suffering Wolf-Hirschhorn syndrome; it encodes a homolog of the yeast protein Mdm38 that is involved in mitochondrial morphology. Here, we describe the LETM1-mediated regulation of the mitochondrial volume and its interaction with the mitochondrial AAA-ATPase BCS1L that is responsible for three different human disorders. LETM1 is a mitochondrial inner-membrane protein with a large domain extruding to the matrix. The LETM1 homolog LETM2 is a mitochondrial protein that is expressed preferentially in testis and sperm. LETM1 downregulation caused mitochondrial swelling and cristae disorganization, but seemed to have little effect on membrane fusion and fission. Formation of the respiratory-chain complex was impaired by LETM1 knockdown. Cells lacking mitochondrial DNA lost active respiratory chains but maintained mitochondrial tubular networks, indicating that mitochondrial swelling caused by LETM1 knockdown is not caused by the disassembly of the respiratory chains. LETM1 was co-precipitated with BCS1L and formation of the LETM1 complex depended on BCS1L levels, suggesting that BCS1L stimulates the assembly of the LETM1 complex. BCS1L knockdown caused disassembly of the respiratory chains as well as LETM1 downregulation and induced distinct changes in mitochondrial morphology.

To better understand the role of human equilibrative (hENTs) and concentrative (hCNTs) nucleoside transporters in physiology and pharmacology, we investigated the regional, cellular, and spatial distribution of two hCNTs (hCNT1 and hCNT2) and two hENTs (hENT1 and hENT2) in four human tissues. Using in situ hybridization and immunohistochemical techniques, we found that the duodenum expressed hCNT1 and hCNT2 mRNAs in enterocytes and hENT1 and hENT2 mRNAs in crypt cells. In these cells, the hCNT and hENT proteins were predominantly localized in the apical and lateral membrane, respectively. Hepatocytes expressed higher levels of mRNAs of hENT1, hCNT1, and hENT2 than of hCNT2 and expressed all these proteins at hepatocyte cell borders and in the cytoplasm. While the kidney expressed hCNT1 and hCNT2 mRNAs in the proximal tubules, hENT1 and hENT2 mRNAs were present in the distal tubules, glomeruli, endothelial cells, and vascular smooth muscle cells. Proximal tubules adjacent to corticomedullary junctions expressed hENT1, hCNT1, and hCNT2 mRNA. Immunolocalization studies revealed predominant localization of hCNTs in the brush-border membrane of the proximal tubular epithelial cells and hENTs in the basolateral membrane of the distal tubular epithelial cells. Chorionic villi sections of human term placenta expressed mRNAs and proteins for hENT1 and hENT2 but only mRNA for hCNT2. Immunolocalization studies showed presence of hENT1 in the brush-border membrane of the syncytiotrophoblasts. These data are critical for a better understanding of the role of nucleoside transporters in the physiological and pharmacological effects of nucleosides and nucleoside drugs, respectively.

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells.

Age-associated accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for the age-associated mitochondrial respiration defects found in elderly human subjects. We carried out reprogramming of human fibroblast lines derived from elderly subjects by generating their induced pluripotent stem cells (iPSCs), and examined another possibility, namely that these aging phenotypes are controlled not by mutations but by epigenetic regulation. Here, we show that reprogramming of elderly fibroblasts restores age-associated mitochondrial respiration defects, indicating that these aging phenotypes are reversible and are similar to differentiation phenotypes in that both are controlled by epigenetic regulation, not by mutations in either the nuclear or the mitochondrial genome. Microarray screening revealed that epigenetic downregulation of the nuclear-coded GCAT gene, which is involved in glycine production in mitochondria, is partly responsible for these aging phenotypes. Treatment of elderly fibroblasts with glycine effectively prevented the expression of these aging phenotypes.