Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels.

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.

The LXR agonist TO901317 selectively lowers hippocampal Abeta42 and improves memory in the Tg2576 mouse model of Alzheimer's disease.

Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein to Abeta peptide. Moreover, cholesterol-rich apoE-containing lipoproteins may also promote Abeta clearance. Agonists of the liver X receptor (LXR) transcriptionally induce genes involved in intracellular lipid efflux and transport, including apoE. Thus, LXR agonists have the potential to both inhibit APP processing and promote Abeta clearance. Here we show that LXR agonist, TO901317, increased hippocampal ABCA1 and apoE and decreased Abeta42 levels in APP transgenic mice. TO901317 had no significant effects on levels of Abeta40, full length APP, or the APP processing products. Next, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the contextual memory deficit in these mice. These data demonstrate that LXR agonists do not directly inhibit APP processing but rather facilitate the clearance of Abeta42 and may represent a novel therapeutic approach to Alzheimer's disease.

Group I metabotropic glutamate receptors, mGlu1a and mGlu5a, couple to cyclic AMP response element binding protein (CREB) through a common Ca2+ - and protein kinase C-dependent pathway.

Coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the cAMP response element binding protein (CREB) has been studied in Chinese hamster ovary cell lines where receptor expression is under the control of an inducible promoter. Both receptors stimulate CREB phosphorylation with similar time courses, and agonist potency was also comparable between the two receptors. Stimulation of cells in Ca(2+)-free medium containing EGTA (100 microm), with or without the additional depletion of intracellular stores, caused marked decreases in agonist-mediated responses in both cell lines. Down-regulation of protein kinase C (PKC) activity by phorbol ester treatment, or treatment with the broad spectrum PKC inhibitor Ro 31-8220, partially attenuated both mGlu1a and mGlu5a receptor-mediated responses. Furthermore, stimulation of cells in the absence of extracellular Ca(2+) following prior PKC down-regulation resulted in additive inhibitory effects. The involvement of extracellular signal-regulated kinases (ERK1/2), Ca(2+)/calmodulin or Ca(2+)/calmodulin-dependent protein kinases was assessed using pharmacological inhibitors. Results indicated that coupling of the group I mGlu receptors to CREB phosphorylation occurs independently of these pathways. Thus, although the [Ca(2+)](i) signatures activated by these mGlu receptors differ, they couple to CREB with comparable potency and recruit similar downstream components to execute CREB phosphorylation.

The PSD95-nNOS interface: a target for inhibition of excitotoxic p38 stress-activated protein kinase activation and cell death.

The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress-generated NO and activation of p38, and the significance of the PSD95-nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95-nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95-nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.

Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models.

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.