In vivo analysis of undocked connexin43 gap junction hemichannels in ovarian granulosa cells.

Connexin43 (Cx43, encoded by Gja1) is required for ovarian follicle development in the mouse. It is strongly expressed in granulosa cells, in which it forms intercellular gap junction channels that couple the cells metabolically. However, recent evidence indicates that undocked gap junction hemichannels can also have physiological roles such as mediating the release of small messenger molecules, including ATP. In this study, the presence of undocked Cx43 hemichannels in granulosa cells was revealed by dye uptake induced either by mechanical stimulation or by the reduction of extracellular divalent cations, both of which are known triggers for hemichannel opening. ATP release was also detected, and could be abolished by connexin-channel blockers. None of these putative hemichannel-mediated activities were detected in Cx43-deficient granulosa cells. Therefore, we hypothesized that hemichannels account for the essential role of Cx43 in folliculogenesis. To test this, a Cx43 mutant lacking the conserved cysteines on the extracellular loops (cys-less Cx43), reported to form hemichannels but not intercellular channels, was retrovirally expressed in Cx43-deficient granulosa cells. The infected cells were then combined with wild-type oocytes to make reaggregated ovaries, which were grafted into host kidneys. Although re-introduction of wild-type Cx43 rescued folliculogenesis, introduction of cys-less Cx43 did not. Therefore, although Cx43 gap junction hemichannels might play a role in ovarian folliculogenesis, their contribution does not supplant the need for intercellular gap junction channels.

The cholesterol transporter ABCG1 modulates the subcellular distribution and proteolytic processing of beta-amyloid precursor protein.

Although intracellular cholesterol levels are known to influence the proteolysis of beta-amyloid precursor protein (APP), the effect of specific genes that regulate cholesterol metabolism on APP processing remains poorly understood. The cholesterol transporter ABCG1 facilitates cholesterol efflux to HDL and is expressed in brain. Notably, the human ABCG1 gene maps to chromosome 21q22.3, and individuals with Down syndrome (DS) typically manifest with Alzheimer's disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-beta protein (Abeta) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)alpha and sAPPbeta secretion and display increased cell surface-associated APP. These results suggest that ABCG1 increases the availability of APP as a secretase substrate for both the amyloidogenic and nonamyloidogenic pathways. In vivo, ABCG1 mRNA levels are 2-fold more abundant in DS brain compared with age- and sex-matched normal controls. Finally, both Abeta and sAPPalpha levels are increased in DS cortex relative to normal controls. These findings suggest that altered cholesterol metabolism and APP trafficking mediated by ABCG1 may contribute to the accelerated onset of AD neuropathology in DS.

Elevated plasma triglyceride levels precede amyloid deposition in Alzheimer's disease mouse models with abundant A beta in plasma.

Dietary or pharmacological manipulation of plasma lipids markedly influences amyloid deposition in animal models of Alzheimer's Disease (AD). However, it is not known whether baseline plasma lipids in AD models differ from wild-type littermates throughout the natural history of disease. To address this question, we measured plasma total cholesterol and triglyceride levels over time in three transgenic AD mouse models in the absence of dietary or pharmacological treatments. Total cholesterol levels were not significantly different between transgenic and wild-type mice during the development of AD neuropathology in all models tested. In contrast, elevated very-low-density lipoprotein (VLDL) triglyceride levels preceded amyloid deposition in two AD models with abundant plasma A beta. Elevated triglycerides were not accompanied by increased inflammatory markers nor decreased lipase activity, but were associated with a significant 30% increase in VLDL-triglyceride secretion rate. Our results suggest that the presence of A beta in plasma may affect peripheral lipid metabolism early in AD pathogenesis.

The absence of ABCA1 decreases soluble ApoE levels but does not diminish amyloid deposition in two murine models of Alzheimer disease.

ABCA1, a cholesterol transporter expressed in the brain, has been shown recently to be required to maintain normal apoE levels and lipidation in the central nervous system. In addition, ABCA1 has been reported to modulate beta-amyloid (Abeta) production in vitro. These observations raise the possibility that ABCA1 may play a role in the pathogenesis of Alzheimer disease. Here we report that the deficiency of ABCA1 does not affect soluble or guanidine-extractable Abeta levels in Tg-SwDI/B or amyloid precursor protein/presenilin 1 (APP/PS1) mice, but rather is associated with a dramatic reduction in soluble apoE levels in brain. Although this reduction in apoE was expected to reduce the amyloid burden in vivo, we observed that the parenchymal and vascular amyloid load was increased in Tg-SwDI/B animals and was not diminished in APP/PS1 mice. Furthermore, we observed an increase in the proportion of apoE retained in the insoluble fraction, particularly in the APP/PS1 model. These data suggested that ABCA1-mediated effects on apoE levels and lipidation influenced amyloidogenesis in vivo.