Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.University of California, Davis. School of Veterinary Medicine. Center for Companion Animal Healt

Copper-Dependent Trafficking of the Ctr4-Ctr5 Copper Transporting Complex

In Schizosaccharomyces pombe, copper uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Copper-induced differential subcellular localization may play a critical role with respect to fine tuning the number of Ctr4 and Ctr5 molecules at the cell surface.We have developed a bimolecular fluorescence complementation (BiFC) assay to analyze protein-protein interactions in vivo in S. pombe. The assay is based on the observation that N- and C-terminal subfragments of the Venus fluorescent protein can reconstitute a functional fluorophore only when they are brought into tight contact. Wild-type copies of the ctr4(+) and ctr5(+) genes were inserted downstream of and in-frame with the nonfluorescent C-terminal (VC) and N-terminal (VN) coding fragments of Venus, respectively. Co-expression of Ctr4-VC and Ctr5-VN fusion proteins allowed their detection at the plasma membrane of copper-limited cells. Similarly, cells co-expressing Ctr4-VN and Ctr4-VC in the presence of Ctr5-Myc(12) displayed a fluorescence signal at the plasma membrane. In contrast, Ctr5-VN and Ctr5-VC co-expressed in the presence of Ctr4-Flag(2) failed to be visualized at the plasma membrane, suggesting a requirement for a combination of two Ctr4 molecules with one Ctr5 molecule. We found that plasma membrane-located Ctr4-VC-Ctr5-VN fluorescent complexes were internalized when the cells were exposed to high levels of copper. The copper-induced internalization of Ctr4-VC-Ctr5-VN complexes was not dependent on de novo protein synthesis. When cells were transferred back from high to low copper levels, there was reappearance of the BiFC fluorescent signal at the plasma membrane.These findings reveal a copper-dependent internalization and recycling of the heteromeric Ctr4-Ctr5 complex as a function of copper availability

SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome

Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease

The Level of ALR is Regulated by the Quantity of Mitochondrial DNA.

Augmenter of liver regeneration (ALR) contributes to mitochondrial biogenesis, maintenance and to the physiological operation of mitochondria. The depletion of ALR has been widely studied and had serious consequences on the mitochondrial functions. However the inverse direction, the effect of the depletion of mitochondrial electron transfer chain and mtDNA on ALR expression has not been investigated yet. Thus mtDNA depleted, rho0 cell line was prepared to investigate the role of mitochondrial electron transfer chain and mtDNA on ALR expression. The depletion of mtDNA has not caused any difference at mRNA level, but at protein level the expression of ALR has been markedly increased. The regulatory role of ATP and ROS levels could be ruled out because the treatment of the parental cell line with different respiratory inhibitors and uncoupling agent could not provoke any changes in the protein level of ALR. The effect of mtDNA depletion on the protein level of ALR has been proved not to be liver specific, since the phenomenon could be observed in the case of two other, non-hepatic cell lines. It seems the level of mtDNA and/or its products may have regulatory role on the protein level of ALR. The up-regulation of ALR can be a part of the adaptive response in rho0 cells that preserves the structural integrity and the transmembrane potential despite the absence of protein components encoded by the mtDNA