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    • Although mature fibrils formed from A aggregates have long

    2023-01-10

    Although mature fibrils formed from Aβ KH CB19 have long been thought to be the main neurodegenerative agents in AD, recent evidence suggests that soluble multimers of these pathogenic proteins, referred to here as “oligomers,” may initiate the synaptic and neuronal dysfunction associated with AD and the other protein-misfolding diseases (the oligomer hypothesis) [25], [27], [32], [40], [42], [48], [49]. In addition, multiple lines of evidence suggest that a pathogenic change in tau may be mediated by low molecular weight order (low-n) oligomers of Aβ, such as dimers and trimers [15], [47]. If so, therapeutic agents must target the assembly or neurotoxic activity of these structures for highest efficacy. In the present study, we examined the effects of CSZ on aggregation of Aβ1-40 and Aβ1-42in vitro, including oligomer formation, using the thioflavin binding assay, photo-induced cross-linking of unmodified proteins (PICUP), and electron microscopy (EM).
    Materials and methods
    Results
    Discussion In the present study, we investigated the effects of the selective PDE3 inhibitor CSZ on the aggregation, including oligomer formation of fresh Aβ1-40 and Aβ1-42 by ThT, PICUP, and EM assays in vitro. Although CSZ significantly inhibited both Aβ1-40 and Aβ1-42 aggregation, its inhibitory effect on oligomerization was stronger than that on fibrillization. Alternatively, CSZ did not inhibit the oligomerization of a control peptide, GST. For monitoring temporal changes in β-sheet content in samples of Aβ1-40 and Aβ1-42 in the absence or presence of CSZ, we used the ThT assay. Although β-sheet formation generally correlates with fibril formation during peptide assembly [20], [21], [23], we previously reported that low n-order oligomers such as PICUP-derived oligomers began to exhibit β-sheet content at the dimer stage, whereas they did not exhibit a ThT fluorescence increase [27]. Although it was once thought that intermediate aggregates of Aβ are positioned on the on-pathway from monomer to mature fibrils, some aggregates such as Aβ-derived diffusible ligands and amylospheroids are on the off-pathway and are more toxic [12], [17], [25], [36]. Very recently, we reported that high n-order oligomers such as protofibrils were also on the off-pathway using combined high-speed atomic force microscopy and ThT assay [50]. Thus, one explanation for the greater inhibitory potency of CSZ against Aβ1-40 and Aβ1-42 oligomerization compared to fibrillization is that low n-order oligomers generated by PICUP are on the off-pathway (Fig. 6). We previously reported that several antioxidant compounds such as wine-related polyphenols, curcumin, and rosmarinic acid have inhibitory effects on Aβ and α-synuclein (αS) aggregation including oligomerization [28], [29], [31], [33], [46]. It was suggested that a phenolic compound with hydoxyl groups, especially in the ortho position, may be a good antioxidant [51]. From the results of curcumin and rosmarinic acid, we also speculated that the orthoquinone ring of these molecules may be suitable for specifically binding to free Aβ/αS and thereby inhibiting aggregation [28], [31]. Similarly, it was reported that the orthoquinone ring of dopamine may covalently bind to monomeric Aβ and αS, thus inhibiting further stages of Aβ and αS aggregation [4]. Although CSZ has no orthoquinone ring, the quinolone ring has antioxidant activity (Fig. 1) [34], which may be related with the binding to Aβ and/or the inhibition of Aβ aggregation. Recent research reported that repeated treatment with CSZ completely prevented the accumulation of lipid peroxidation induced by Aβ25–35 peptide and attenuated cognitive impairments in mice [11]. Similarly, CSZ reduced infarct volumes and Nox 2-positive neurons in mice with permanent focal ischemia [43] and inhibited ROS production via inhibition of Nox in murine macrophages [52]. It was reported that CSZ protected against Aβ1-40-induced cellular toxicity and preserved neurite elongation through CK2α activation by downregulating p53 phosphorylation and upregulating GSK-3β phosphorylation [18]. They also reported that CSZ stimulated the clearance of Aβ1-42 and C-terminal fragments (CTF) of amyloid precursor protein by upregulation of autophagy via SIRT activation in neuronal cells, thereby reducing Aβ-induced neurotoxicity [19]. Very recently, we reported that pretreatment with CSZ significantly suppressed Aβ1-42-induced oxidative stress and cytotoxicity in SH-SY5Y cells by suppression of NOX activity and also by providing protection against mitochondrial dysfunction [24]. The underlying mechanisms behind the protective effects of CSZ on Aβ1-42-induced cytotoxicity may include reducing ROS production and increasing cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation through extracellular signal-regulated kinase (ERK) activity [24].