Modulation of ABCA1 by an LXR agonist reduces ß-amyloid levels and improves outcome after traumatic brain injury.

Traumatic brain injury (TBI) increases brain beta-amyloid (Aß) in humans and animals. Although the role of Aß in the injury cascade is unknown, multiple preclinical studies have demonstrated a correlation between reduced Aß and improved outcome. Therefore, therapeutic strategies that enhance Aß clearance may be beneficial after TBI. Increased levels of ATP-binding cassette A1 (ABCA1) transporters can enhance Aß clearance through an apolipoprotein E (apoE)-mediated pathway. By measuring Aß and ABCA1 after experimental TBI in C57BL/6J mice, we found that Aß peaked early after injury (1-3 days), whereas ABCA1 had a delayed response (beginning at 3 days). As ABCA1 levels increased, Aß levels returned to baseline levels-consistent with the known role of ABCA1 in Aß clearance. To test if enhancing ABCA1 levels could block TBI-induced Aß, we treated TBI mice with the liver X-receptor (LXR) agonist T0901317. Pre- and post-injury treatment increased ABCA1 levels at 24 h post-injury, and reduced the TBI-induced increase in Aß. This reduction in Aß was not due to decreased amyloid precursor protein processing, or a shift in the solubility of Aß, indicating enhanced clearance. T0901317 also limited motor coordination deficits in injured mice and reduced brain lesion volume. These data indicate that activation of LXR can reduce Aß accumulation after TBI, and is accompanied by improved functional recovery.

Regulation of central nervous system cholesterol homeostasis by the liver X receptor agonist TO-901317.

The nuclear oxysterol receptors known as liver X receptors (LXR) are responsible for regulating genes involved in cholesterol homeostasis. Two subtypes exist, LXR-alpha and LXR-beta, both of which are expressed in the central nervous system (CNS). Activation of LXR causes an increase in mechanisms involved in cholesterol efflux, including ATP-binding cassette transporters (ABC)-A1 and ABC-G1. Although LXR agonists have been found to induce gene expression for ABC-A1 and ABC-G1 in the CNS, no functional response has been recorded. In this study, we show that an LXR agonist (TO-901317) increases protein levels of the cholesterol transporters ABC-A1 and ABC-G1, as well as the cholesterol chaperone protein apolipoprotein E (apoE). These changes are associated with a decrease in cholesterol levels from isolated cortical synaptosomal plasma membranes. LXR activation occurs in response to increased cholesterol levels in cells. However, while data exists on how LXR alter cholesterol efflux, there is no data on whether these receptors alter production of cholesterol. We found that TO-901317 increases HMG-CoA reductase activity, the rate-limiting step of cholesterol production in vivo. This finding was duplicated in an in vitro system. Although high concentrations (>1 microM) of TO-901317 were required to induce HMG-CoA reductase activity, these changes only occurred in the presence of apolipoprotein AI, suggesting that cholesterol efflux is required for this effect to occur. These data show that the LXR agonist TO-901317 is capable of reducing cholesterol in neurons of the CNS.

Cholesterol independent effect of LXR agonist TO-901317 on gamma-secretase.

The balance of intracellular cholesterol has proven to be critical to the production of beta-amyloid (A beta). Reducing cholesterol in vitro leads to decreased production of A beta, whereas an increase in cellular cholesterol induces A beta production. Liver X Receptor (LXR) agonists are known to increase cholesterol efflux from cells, but there are conflicting reports as to the effects of these agonists on A beta production. We therefore examined the effects of efflux-inducing agents on A beta production in vitro. We used methyl-beta-cyclodextrin and an LXR agonist (TO-901317) to induce cholesterol efflux and studied the resulting A beta production in a stable amyloid precursor protein (APP) -transfected cell line. When cholesterol efflux was induced with methyl-beta-cyclodextrin there was a >60% decrease in A beta(40) and A beta(42) production. However, while activation of LXR using TO-901317-induced cholesterol efflux in the presence of a cholesterol acceptor, no changes in A beta levels were recorded. When cells were incubated with TO-901317 above the concentration required for maximal cholesterol efflux, there was a 150% increase in A beta(42) levels. The absence of a cholesterol acceptor from the culture media (preventing cholesterol efflux) did not blunt this increase in A beta(42), suggesting that the effects of TO-901317 on A beta(42) are efflux independent. These results were confirmed in APP stably transfected human H4 cells, which revealed in addition to a 200% increase in A beta(42) levels, a concomitant 80% reduction in A beta(38). A cell-free gamma-secretase assay confirmed that TO-901317 can directly alter gamma-secretase activity. These data demonstrate that TO-901317 can directly modulate the site of cleavage of APP by gamma-secretase in vitro.

The effects of ABCA1 on cholesterol efflux and Abeta levels in vitro and in vivo.

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of beta-amyloid (Abeta), a peptide that is overproduced in Alzheimer's disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Abeta have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Abeta levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO-901317) on wild-type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Abeta levels. We then examined endogenous Abeta levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Abeta levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO-901317, caused no change in Abeta levels. However, when efflux capability was restored, TO-901317 reduced Abeta levels. These data show that promoting cholesterol efflux is a viable target for Abeta reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Abeta in vitro or in vivo.

Gadd45a regulates matrix metalloproteinases by suppressing DeltaNp63alpha and beta-catenin via p38 MAP kinase and APC complex activation.

The p53-regulated growth arrest and DNA damage-inducible gene product Gadd45a has been recently identified as a key factor protecting the epidermis against ultraviolet radiation (UVR)-induced skin tumors by activating p53 via the stress mitogen-activated protein kinase (MAPK) signaling pathway. Herein we identify Gadd45a as an important negative regulator of two oncogenes commonly over-expressed in epithelial tumors: the p53 homologue DeltaNp63alpha and beta-catenin. DeltaNp63alpha is one of the several p63 isoforms and is the predominant species expressed in basal epidermal keratinocytes. DeltaNp63alpha lacks the N-terminal transactivation domain and behaves as a dominant-negative factor blocking expression of several p53-effector genes. DeltaNp63alpha also associates with and blocks activation of the adenomatous polyposis coli (APC) destruction complex that targets free cytoplasmic beta-catenin for degradation. While most beta-catenin protein is localized to the cell membrane and is involved in cell-cell adhesion, accumulation of free cytoplasmic beta-catenin will translocate into the nucleus where it functions in a bipartite transcription factor complex, whose targets include invasion and metastasis promoting endopeptidases, matrix metalloproteinases (MMP). We show that Gadd45a not only directly associates with two components of the APC complex, namely protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta) but also promotes GSK3beta dephosphorylation at Ser9, which is essential for GSK3beta activation, and resultant activation of the APC destruction complex. We demonstrate that lack of Gadd45a not only prevents DeltaNp63alpha suppression and GSK3beta dephosphorylation but also prevents free cytoplasmic beta-catenin degradation after UV irradiation. The inability of Gadd45a-null keratinocytes to suppress beta-catenin may contribute to the resulting observation of increased MMP expression and activity along with significantly faster keratinocyte migration in Matrigel in vitro and accelerated wound closure in vivo. Furthermore, epidermal keratinocytes treated with p38 MAPK inhibitors, both in vivo and in vitro, behave very similarly to Gadd45a-null keratinocytes after UVR. Similarly, Trp53-null mice are unable to attenuate DeltaNp63alpha expression in epidermal keratinocytes after such stress. These findings demonstrate a dependence on Gadd45a-mediated p38 MAPK and p53 activation for proper modulation of DeltaNp63alpha, GSK3beta, and beta-catenin after irradiation. Taken together, our results indicate that Gadd45a is able to repress DeltaNp63alpha, beta-catenin, and consequently MMP expression by two means: by maintaining UVR-induced p38 MAPK and p53 activation and also by associating with the APC complex. This implicates Gadd45a in the negative regulation of cell migration, and invasion.

Gadd45a protects against UV irradiation-induced skin tumors, and promotes apoptosis and stress signaling via MAPK and p53.

Skin cancer is the most frequent form of malignancy in the world, and UV radiation is the primary environmental carcinogen responsible for its development. Herein we demonstrate that Gadd45a is a critical factor protecting the epidermis against UV radiation-induced tumorigenesis by promoting damaged keratinocytes to undergo apoptosis and/or cell cycle arrest, two crucial events that prevent the expansion of mutant or deregulated cells. Whereas Gadd45a has been implicated in cell cycle arrest, apoptosis, and DNA repair, to determine the physiological function of endogenous Gadd45a after genotoxic stress, the skin of Gadd45a-null mice was targeted with UV radiation. We report that Gadd45a induces apoptosis and cell cycle arrest by maintaining p38 and c-JNK MAPK activation in keratinocytes. The absence of Gadd45a results in loss of sustained p38/JNK MAPK activity beyond 15-30 min after UV radiation that leads to inadequate p53 activation and loss of normal activation of G(1) and G(2) checkpoints. Moreover, loss of Gadd45a dramatically reduces UV-induced apoptotic keratinocytes, "sunburn cells." Consequently, Gadd45a-null mice are more prone to tumors relative to wild-type mice. Therefore, we conclude that Gadd45a, like p53, is a key component protecting skin against UV-induced tumors.