Breed distribution of the nt230(del4) MDR1 mutation in dogs.

A 4-bp deletion mutation associated with multiple drug sensitivity exists in the canine multidrug resistance (MDR1) gene. This mutation has been detected in more than 10 purebred dog breeds as well as in mixed breed dogs. To evaluate the breed distribution of this mutation in Germany, 7378 dogs were screened, including 6999 purebred and 379 mixed breed dogs. The study included dog breeds that show close genetic relationship or share breeding history with one of the predisposed breeds but in which the occurrence of the MDR1 mutation has not been reported. The breeds comprised Bearded Collies, Anatolian Shepherd Dog, Greyhound, Belgian Tervuren, Kelpie, Borzoi, Australian Cattle Dog and the Irish Wolfhound. The MDR1 mutation was not detected is any of these breeds, although it was found as expected in the Collie, Longhaired Whippet, Shetland Sheepdog, Miniature Australian Shepherd, Australian Shepherd, Wäller, White Swiss Shepherd, Old English Sheepdog and Border Collie with varying allelic frequencies for the mutant MDR1 allele of 59%, 45%, 30%, 24%, 22%, 17%, 14%, 4% and 1%, respectively. Allelic frequencies of 8% and 2% were determined in herding breed mixes and unclassified mixed breeds, respectively. Because of its widespread breed distribution and occurrence in many mixed breed dogs, it is difficult for veterinarians and dog owners to recognise whether MDR1-related drug sensitivity is relevant for an individual animal. This study provides a comprehensive overview of all affected dog breeds and many dog breeds that are probably unaffected on the basis of ∼15,000 worldwide MDR1 genotyping data.

Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease.

Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY-lactosylceramide and -galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol-yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

Process outgrowth of oligodendrocytes is promoted by interaction of fyn kinase with the cytoskeletal protein tau.

Fyn kinase plays an important role during myelination and has been shown to promote morphological differentiation of cultured oligodendrocytes. We analyzed the downstream targets of Fyn kinase in oligodendrocytes. Because process outgrowth and wrapping of axons involve cytoskeletal rearrangement, we focused on cytoskeletal proteins linked to Fyn. Here we demonstrate that Fyn binds to the cytoskeletal proteins Tau and alpha-Tubulin in oligodendrocytes. Tau interacts with the Fyn SH3 domain whereas alpha-Tubulin binds to the Fyn SH2 and SH3 domains. To study the function of the Fyn-Tau interaction in oligodendrocytes, we designed a Tau deletion mutant that would compete with endogenous Tau-Fyn binding in transfected cells. The mutant Tau protein binds to the Fyn SH3 domain but lacks the microtubuli interaction domain and thus cannot bind to microtubuli. In the presence of the mutant Tau protein, a reduction of the process number and process length in oligodendroglial cells was observed. This effect is likely to be caused by interference with the Fyn-Tau-microtubuli cascade rather than inactivation of the kinase, because Fyn bound to the mutant Tau retains activity. A similar inhibition of process outgrowth was observed when oliogodendroglial cells were cultured in the presence of Fumonisin B1, an inhibitor of sphingolipid synthesis that prevents the formation of rafts. Because ligation of the cell adhesion molecule F3 on oligodendrocytes leads to activation of Fyn kinase localized in rafts, these findings suggest that recruitment of Tau and Tubulin to activated Fyn kinase in rafts is an important step in the initiation of myelination.