The loss of synaptic proteins such as synaptophysin

The loss of synaptic proteins such as synaptophysin from the Necrosulfonamide is indicative of synapse degeneration and provided a good correlate of the degree of dementia in AD [12], [13], [14]. Consequently, the loss of synaptic proteins from cultured primary neurons incubated with Aβ provides a useful in vitro model in which to investigate AD-related synapse damage [15]. Synapse density in cultured neurons was measured by quantification of the amounts of synaptophysin and cysteine string protein (CSP) [15]. Picomolar concentrations of soluble Aβ caused the loss of synaptophysin and CSP from cultured neurons [15] and impaired memory formation in animal models [10], [16]. Although many studies implicate cholesterol concentrations as a major factor that regulates Aβ production, as reviewed by Chang and colleagues [17], the effects of cholesterol depletion on Aβ production remain controversial. While initial studies demonstrated that cholesterol depletion reduced the production of Aβ [18], another study reported it increased Aβ concentrations [19]. However, the conventional cholesterol synthesis inhibitors (3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) used to deplete cells of cholesterol also block the production of isoprenoids, which also affect Aβ concentrations [20], [21]. Consequently it is not clear whether the effects of these drugs on Aβ concentrations are mediated by their effects upon cholesterol, or upon isoprenoid concentrations. In addition, previous studies have not differentiated the effects of cholesterol depletion upon the type of Aβ produced (toxic/non-toxic). Here we show that treatment of 7PA2 cells with squalestatin, a squalene synthetase inhibitor that inhibits cholesterol synthesis without affecting the production of isoprenoids [22], reduced the release of synaptotoxic Aβ; conditioned media (CM) from these cells did not cause synapse degeneration when added to cultured neurons. Surprisingly, squalestatin had only a small affect upon the total concentrations of Aβ42 in CM, rather it changed the ratio of Aβ oligomers to Aβ monomers. Treatment reduced concentrations of the synaptotoxic Aβ oligomers and increased concentrations of neuroprotective Aβ monomers [23]. This study also identified other drugs that affected the type of Aβ released; the release of soluble Aβ oligomers was reduced by treating 7PA2 cells with either phospholipase A2 (PLA2) inhibitors, platelet-activating factor (PAF) antagonists or cholesterol ester hydrolase (CEH) inhibitors. The observation that PAF activated CEHs, resulting in increased cholesterol concentrations within the endoplasmic reticulum (ER), led to the hypothesis that PAF-induced activation of CEH releases cholesterol that consequently affected the production of Aβ oligomers.
Methods
Results
Discussion Although many studies have examined the factors that affect Aβ production this is the first study that we are aware of that differentiated between toxic and non-toxic forms of Aβ. The release of biologically active forms of Aβ by 7PA2 cells was examined using synapse damage in cultured neurons as a model. The synapse damage caused by 7PA2-CM was comparable to that caused by soluble brain extracts from AD patients when standardized on their Aβ42 content. In addition, the synapse damage caused by both 7PA2-CM and brain extracts (15) were Aβ-dependent, suggesting that 7PA2 cells release synaptotoxic forms of Aβ similar to the Aβ oligomers found in brains of AD patients. The 2 key findings of this study were firstly, that the release of synaptotoxic Aβ oligomers is controlled by pathways sensitive to squalestatin, PLA2 inhibitors, PAF antagonists and CEH inhibitors. All these compounds affected either cholesterol concentrations or cholesterol distribution within cell membranes. Secondly, that treated cells released increased concentrations of Aβ monomers and that these monomeric forms of Aβ protected neurons against the synaptotoxic Aβ oligomers.