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The neurotoxicity of amyloid-β protein (Aβ) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Aβ aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Aβ, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Aβ have been identified. We have investigated the toxicity induced by intracellular Aβ in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Aβ (Aβ-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Aβ-OCs. In aqueous solution, CP2 attenuates Aβ oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Aβ toxicities within both intracellular and extracellular sites.
Studies of amyloid-β (Aβ) toxicity using cell culture models have mainly focused on the consequences of applying synthetic Aβ peptides extracellularly to the culture media and have contributed tremendously to our understanding of Alzheimer's disease (AD; Pike et al. 1993; Lorenzo and Yankner 1994). Recently, a growing body of evidence suggests that the intraneuronal accumulation of Aβ might also be toxic and might be the first step of the amyloid cascade leading to AD (Wirths et al. 2004; Gouras et al. 2005). Accumulation of Aβ in abnormal endosomes is associated with the earliest Aβ elevations in AD (Cataldo et al. 2004). Intracellular Aβ was found to be at least 100 000 times more toxic than extracellular Aβ (Kienlen-Campard et al. 2002; Zhang et al. 2002). The intraneuronal oligomeric form of Aβ has been linked to neuritic damage and synaptic alterations (Takahashi et al. 2004; Almeida et al. 2005). Studies of several transgenic mouse models also demonstrated early intraneuronal Aβ deposition before the first appearance of amyloid plaques or behavioral deficits (Shie et al. 2003; Casas et al. 2004; Billings et al. 2005). Intraneuronal Aβ was correlated with the earliest behavioral deficits in a line of transgenic mice (Billings et al. 2005). Taken together, these data point out possible therapeutic benefits from cell permeable compounds that target intraneuronal Aβ (Walsh et al. 2002).
A major obstacle has been the paucity of cell culture models showing a convincing link between intracellular Aβ and cell death, which would be simple enough to be used as a screening tool for potentially therapeutic compounds. Established models relied on long-term viral expression of the amyloid-β precursor protein (APP) in rat cortical neurons (Kienlen-Campard et al. 2002) and micro-injection of synthetic Aβ peptides or an Aβ-expressing cDNA into human cortical neurons (Zhang et al. 2002). A recent model using primary neurons from Tg2576 APP transgenic mice shows Aβ oligomers accumulating in neuronal processes (Takahashi et al. 2004). However, the preparation of this model is relatively time-consuming and the toxicity requires detection by electron microscopy.
By contrast, the MC65 cell line can be conveniently used to screen bioactive compounds (Sopher et al. 1994; Jin et al. 2002; Maezawa et al. 2004; Woltjer et al. 2005). MC65 is a line of human neuroblastoma that conditionally expresses C99, a 99-residue carboxyl terminal fragment derived from the β-secretase cleavage of APP. C99 is subsequently cleaved by γ-secretase to generate Aβ. Following transgene induction, significant loss of cell viability occurs after 68 h. The death is not caused by factors in the media, including secreted Aβ (Sopher et al. 1994). Compared with all the models mentioned above, MC65 cells are easily propagated and the cell toxicity is measured quantitatively by a simple 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. However, the exact intracellular C99-related mechanism leading to cell death is not known.
Previously, we showed that several tricyclic pyrones (TPs) protected MC65 cells from death (Jin et al. 2002; Hua et al. 2003). However, the mechanism of this protection is not known. TPs were first synthesized based on the structures of pyripyropene A, a potent acyl-CoA : cholesterol O-acyltransferase inhibitor (Omura et al. 1993), and arisugacin, a potent acetylcholinesterase inhibitor (Omura et al. 1995; Hua et al. 1997a). Both activities have been shown to affect Aβ production or aggregation (Inestrosa et al. 1996; Hutter-Paier et al. 2004). However, a few TPs protect MC65 cells at concentrations at least 1000-fold lower than those required for acyl-CoA : cholesterol O-acyltransferase and acetylcholinesterase inhibition (Hua et al. 2003), therefore these two activities cannot explain the MC65 protective effect. Here, we demonstrate that a lead TP named CP2 is cell permeable and is able to block Aβ aggregation, effects that may underlie its MC65 protection.
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- Materials and methods
Intracellular aggregation of proteins is increasingly being linked to neurodegenerative processes and occurs in increasing numbers of diseases collectively called conformational diseases. The mechanism by which intracellular Aβ misfolds, aggregates, and becomes toxic to neurons remains poorly understood. Cell models and inhibitor molecules would contribute to our understanding of this mechanism. We demonstrated that MC65 cells could serve as a model for studying the death of neuron-like cells as a result of intracellular Aβ. MC65 cell death was closely associated with and likely dependent upon the intracellular aggregated forms of Aβ. We took advantage of these properties and successfully selected CP2 that was cell permeable and capable of inhibiting intracellular Aβ aggregation into Aβ-OCs and extracellular Aβ aggregation into oligomers. Our results validate the usefulness of the MC65 protection assay for the screening of small molecules with anti-Aβ aggregation properties.
The rescuing effect demonstrated by the γ-secretase inhibitor clearly indicates that MC65 death is dependent on the production of Aβ. Based on our ELISA quantification of secreted Aβ and Aβ in the guanidine extracts of MC65 cells (Jin et al. 2004), the intracellular Aβ, on average, was only approximately 6% of the total Aβ produced. However, probably because of its high focal concentrations, its strong membrane toxicity (Kayed et al. 2004), or the contributions from unknown cellular co-factors, intracellular Aβ is toxic to cells despite its small quantity. Furthermore, the observations that both L-685 458 and CP2 reduced Aβ-OCs, but CP2 did not reduce the overall Aβ level, strongly suggest that Aβ-OCs might be the major cause of MC65 cell death. Although other indirect effects on intracellular Aβ aggregation/toxicity are possible, our data strongly suggest that a direct effect of CP2 on intracellular Aβ aggregation may, at least in part, underlie CP2′s MC65 protective effect.
Previously, we showed that antioxidants fully protect MC65 cells (Sopher et al. 1996; Maezawa et al. 2004). We excluded the possibility that CP2 was an antioxidant because: (i) the cyclic voltammogram revealed that the oxidation potential Ep,a of CP2 was +1.1 V, too high to be considered an antioxidant (de Souza et al. 2003). By comparison, under similar experimental conditions, the Ep,a of ferrocene was +0.5 V. Typical antioxidants, such as flavonoids, have Ep,a values between +0.58 and +0.91 V; (ii) the HT-22 oxidative stress assay (Tan et al. 1998) showed no protective effect by CP2; (iii) CP2 did not protect from cell toxicities as a result of H2O2 treatment or culturing in 95% oxygen. Furthermore, CP2 was not effective in rescuing cell death in apoptotic death models such as serum withdrawal from PC12 cells or nerve growth factor withdrawal from differentiated PC12 cells (data not shown) (Greene 1978). Therefore, it appears that CP2 is not a general antioxidant nor an anti-apoptotic agent.
To our knowledge, CP2 is the first characterized small molecule that inhibits intracellular Aβ aggregation and blocks intracellular Aβ toxicity. Several small molecules, such as azodye derivatives [for example, chrysamine G (CG) and RS-0406], curcumin and DAPH (4,5-dianilinophthalimide), have been characterized based on their ability to inhibit the aggregation of synthetic Aβ peptides in aqueous solution (Blanchard et al. 2004; Walsh et al. 2005; Yang et al. 2005). It remains to be shown whether they block intracellular Aβ aggregation, a process intimately related to membrane/vesicle structures. The azodyes and curcumin share a symmetrical flat structure that allows alignment with the β-pleated sheet (Yang et al. 2005), thus providing their anti-aggregation effect. By comparison, CP2 has an asymmetric structure, while TP17, a flat structure as revealed by X-ray analysis (Hua et al. 1997b), is entirely inactive in our assays. Therefore, CP2 appears to have an anti-aggregation property distinct from that of azodyes and curcumin. Interestingly, we found that CG efficiently attenuated Aβ aggregation in an aqueous solution, similar to CP2. However, neither CG nor curcumin offered MC65 protection (data not shown), despite their previously suggested antioxidation and anti-Aβ effects (Lim et al. 2001; Ishii et al. 2002). Therefore, our data raise an interesting possibility that the mechanisms of aggregation involving vesicle/membrane-associated, cell-produced Aβ would be distinct from those observed with synthetic Aβ in an aqueous solution. It is highly desirable that, in addition to inhibiting the Aβ aggregation in the aqueous solution, an anti-Aβ compound also inhibits the aggregation of membrane/vesicle-associated Aβ and counteracts the deleterious effects of Aβ on the membrane. Our data suggest that CP2 might possess such desirable properties.
Despite their simple core structure, the tricyclic pyrones promise a boundless potential for a variety of novel compounds, which may be fine-tuned to produce leads with desired properties. As demonstrated in our data, small modifications of CP2 can significantly change the biological and biophysical effects of the compounds. Possible modifications of CP2 include functionalization of the methyl group of the pyrone ring, the C7 alkyl side chain, the C9 allylic methylene, and the C10 double bond, to name a few. Knowledge of the structure–activity relationship based on future CP2 derivatives should help elucidate the poorly understood mechanism of intraneuronal Aβ aggregation and toxicity.