Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.     

Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

We isolated and characterized a novelK-Cl cotransporter, KCC3, from human placenta. The deduced proteincontains 1,150 amino acids. KCC3 shares 75-76% identity at theamino acid level with human, pig, rat, and rabbit KCC1 and 67%identity with rat KCC2. KCC3 is 40 and 33% identical to twoCaenorhabditis elegans K-Cl cotransporters and ~20%identical to other members of the cation-chloride cotransporter family(CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Clcotransporter (NCC). Hydropathy analysis indicates a typical KCCtopology with 12 transmembrane domains, a large extracellular loopbetween transmembrane domains 5 and 6 (unique to KCCs), and largeNH₂ and COOH termini. KCC3 is predominantly expressed inkidney, heart, and brain, and is also expressed in skeletal muscle,placenta, lung, liver, and pancreas. KCC3 was localized to chromosome15. KCC3 transiently expressed in human embryonic kidney (HEK)-293cells fulfilled three criteria for increased expression of K-Clcotransport: stimulation of cotransport by swelling, treatment withN-ethylmaleimide, or treatment with staurosporine.