Cholesterol homeostasis has proven to be intimately linked to various aspects of Aβ biology [2
]. In addition to statins, ACAT inhibitors are promising therapeutic strategies for Alzheimer’s disease since both reduce amyloid plaque density in animal models of the disease. It appears that the balance between amyloidogenic and nonamyloidogenic APP processing is especially sensitive to changes in intracellular cholesterol distribution [8
]. As ACAT is a key regulator of the equilibrium between free cholesterol and cholesteryl ester pools, it is not surprising that inhibition of ACAT suppresses proteolytic processing of APP. In this study, we investigated whether a knockdown of ACAT-1 expression by RNAi in cells affects proteolytic processing of APP and production of Aβ. To detect secreted Aβ species in the culture medium we used a model overexpressing human APP751
. It should be noted that we have detected similar suppressive effects of ACAT-1 RNAi on the proteolytic processing of endogenous APP in H4 naïve cells (data not shown). To control the specificity of the RNAi we used a mixture of three siRNA oligonucleotides specific for mouse ACAT-1. Importantly, even in the high 3 μg dose the control siRNA did not display any effects on the key parameters of this study, ACAT-1 expression, FC and CE levels and APP/Aβ levels. As we routinely perform 4-day treatments in cell-based ACAT-inhibitor studies, this study was designed to be a 4-day study for comparability. Importantly, RNAi-induced decrease in ACAT-1 expression to roughly half of the control cell levels resulted in a significant suppression of Aβ production (~40%) despite the rather modest ~22% decrease in cholesteryl ester levels. Both Aβ40
were affected more or less equally. These results are comparable to the previously published results that used ACAT-1 inhibitors [8
]. Interestingly, ACAT-1 RNAi caused a similar although smaller increase in the free cholesterol content of ER membranes as pharmacological ACAT inhibition. This may have important mechanistic implications and require further studies.
As has been previously shown for pharmacological ACAT inhibition [8
], the results presented here suggest that ACAT-1 RNAi affects all of the three α-, β- and γ-secretases as not only Aβ and APP-C99 but also APP-C83 levels were decreased by ACAT-1 RNAi. Although the mechanistic details on how ACAT inhibition modulates APP processing require further studies, it seems that pharmacological ACAT inhibition and ACAT-1 RNAi affect the same or similar pathways in the early secretory pathway that are important for APP metabolism. Recently, the specificity of certain ACAT inhibitors has been questioned [12
]. Thus, this study provides an important, nonpharmacological proof-of-principle confirming that reduction of cellular ACAT activity is a viable and specific approach for modulating Aβ generation.