AD is the most common neurodegenerative disorder characterized by progressive memory impairment and cognitive deficits. The pathology of AD is still unclear; however, several studies have shown that the aggregation of the Aβ peptide in the CNS is an exclusively pathological process involved in AD. Currently, there is no proven medication to cure or prevent the disease progression. Nevertheless, various therapeutic approaches for AD show only relief of symptoms and mostly work on cognitive recovery. However, one of the promising approaches for therapeutic intervention is to use inhibitors for blocking the Aβ peptide aggregation process. Recently, herbal phenolic compounds have been shown to have a therapeutic property for treatment of AD due to their multifaceted action. In this study, we investigated the effectiveness of SA, Gn Rb1, and DMyr on inhibiting the aggregation and toxicity of Aβ40 and Aβ42 using different biochemical and cell-based assays. Our results showed that SA and DMyr inhibit Aβ40 and Aβ42 fibrillation, seeded aggregation, and toxicity. Gn Rb1 did not have any effect on the aggregation or toxicity induced by Aβ40 and Aβ42. Moreover, SA and DMyr were able to disaggregate the preformed fibrils. Overall, these compounds may be used alone or synergistically and could be considered as a lead for designing new compounds that could be used as effective treatment of AD and related disorders.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder caused by gradual loss of cholinergic neurons in limbic and neocortical regions [1]. AD is characterized by the presence of neurofibrillary tangles and neuritic plaques, commonly referred to as senile plaques. Neurofibrillary tangles primarily consist of tau aggregates. Abnormal tau proteins undergo hyperphosphorylation, resulting in their misfolding and subsequent aggregation into insoluble tangles within neurons. These tangles disrupt normal brain cell function, impair neuronal communication, and ultimately lead to neurodegeneration [1]. The plaques primarily consist of 39- to 43-residue Amyloid beta (Aβ) peptides, which are derived from the transmembrane amyloid-beta precursor protein (APP) through endoproteolytic cleavage. APP are cleaved by gamma secretase, resulting in the formation of Aβ fragments. The accumulation of Aβ as plaques leads to neuronal death [2]. This neurodegeneration leads to a depletion in glutamate and acetylcholine neurotransmitters, which correlates with the cognitive impairment and behavioral changes that AD patients experience [3]. Neuropathological studies have shown the buildup of amyloid beta (Aβ) plaque deposits outside of and neurofibrillary tangles in neurons, causing a decrease in cholinergic neurotransmission and cognitive impairment [4]. Currently, the focus of research is the amyloid hypothesis of AD, which states that the accumulation of Aβ outside of neurons is responsible for cognitive impairment and neuronal death [5]. This accumulation triggers an inflammatory response, synaptic dysfunction, and alters neuronal homeostasis, leading to neuronal death and AD [6,7]. Studies have supported the cause of AD due to misfolded aggregates of human Aβ42, rather than more abundant Aβ40 [8,9].
In its native form, Aβ plays a normal physiological role in healthy individuals. However, in AD patients, Aβ expression increases, leading to the formation of aggregates, which are β-sheet structures that are deposited as senile plaques [10,11,12]. Similar to any other amyloid protein and peptides, Aβ undergoes a highly dynamic self-assembly process into amyloid fibrils, resulting in the formation of various intermediates with differences in size, structure, and shape. Recent studies show that the aggregation process proceeds in a nucleation-dependent manner, forming mature fibrils through intermediate stages such as oligomers and protofibrils. Increasing evidence suggests that these prefibrillar soluble oligomers rather than the mature fibrils of Aβ are responsible for neurodegeneration and synaptic dysfunction in AD. Moreover, mature fibrils can indirectly contribute to neuronal damage by binding to and activating microglia [6,13]. Current drugs for AD only provide symptomatic relief and do not prevent or reverse the disease. Therefore, searching for compounds that can inhibit the formation of early Aβ aggregates may be a promising strategy for therapeutic intervention in AD and related disorders [4,14].
Plant-derived natural polyphenols, such as those found in tea, nuts, berries, and cocoa, have been found to have a demonstrated promising effect on the aggregation of Aβ peptides in vitro, owing to their aromatic phenolic structure [15]. Studies have highlighted the neuroprotective properties of specific compounds such as myricetin and ginsenoside Rb1 (GnRb1), which exhibit inhibitory effects on the progression of AD [16,17,18,19]. Furthermore, salvianolic acid B (SA), through its antioxidative and anti-inflammatory effects, has been shown to possess neuroprotective function in an Aβ25–35 peptide-induced mouse model of AD [20]. In addition, Tang et al. demonstrated that SA inhibits Aβ generation by modulating APP processing in SH-SY5Y-APPsw cells [21].
Given the common process of aggregation among amyloid protein, drugs targeting the aggregation process of one disease may exhibit promising effects on others. Previous studies on α-synuclein, a protein implicated in Parkinson’s disease that shares a similar aggregation pattern with Aβ peptides, have shown that Gn Rb1 acts as a potent inhibitor of α-synuclein aggregation and toxicity of [22].
The formation of Aβ plaques resulting from accumulation of Aβ in the brain is widely recognized as the primary pathological hallmark of AD. Consequently, targeting the aggregation process of both Aβ40 and Aβ42 peptides holds considerable promise as a therapeutic strategy for managing the disease. In this study, we investigated the effect of Gn Rb1, SA, and DMyr on the aggregation of Aβ40 and Aβ42, with the aim of evaluating their potential as drug candidates for AD treatment.
Aβ40 and Aβ42 solutions (100 µM) either alone or with SA, Gn Rb1, and DMyr at different molar ratios (Aβ: compounds; 1:1, 1:5, and 1:10) were incubated at 37 °C for 14 days. The aggregation was monitored by the Th-S fluorescence assay at regular time intervals.
We found that both SA and DMyr inhibit the aggregation of Aβ40 and Aβ42 in a concentration-dependent manner. For Aβ42, the inhibition of aggregation was observed as early as 2 days of incubation with DMyr, and when used at a molar excess of 1:10, a complete inhibition was observed at day 4 and day 10 for DMyr and SA, respectively. In contrast, the inhibition effect of SA and DMyr on Aβ40 was most prominent after 10 days of incubation, and a complete inhibition of aggregation was observed in both compounds at ratio 1:10. However, using Gn Rb1, we did not observe any significant effect on the process of aggregation for either Aβ40 or Aβ42.
These findings were further confirmed by electron microscopy. TEM images showed that in the presence of SA or DMyr, Aβ40 and Aβ42 formed less dense, thin, and short fibrils, and small early aggregates, in a concentration-dependent manner, as compared to dense meshes of long fibrils formed by aged Aβ40 and Aβ42 alone. In the presence of Gn Rb1, TEM images were similar to Aβ40 and Aβ42 alone, consistent with the Th-S fluorescence assay.
Next, we measured the levels of Aβ40 and Aβ42 early aggregates formed during the aggregation process. Aliquots of Aβ40 and Aβ42 incubated alone or with different ratios of compounds (Aβ: compounds; 1:1, 1:5, and 1:10) during the aggregation assay were taken and assessed by oligomeric ELISA using Aβ-specific antibody 6 × 10 10. We observed that with the addition of either SA or DMyr, stabilizes the fibrillation, as observed by the constant signal obtained during aggregation; whereas in the control samples, the level of Aβ40 and Aβ42 signal decreased after day 6, depicting the formation of mature fibrils containing a smaller number of exposed epitopes. However, in the Aβ42 sample containing SA at a 1:10 molar ratio, the signal was less than the control. Thus, these data demonstrate that SA and DMyr could stabilize the Aβ40 and Aβ2 early aggregation and SA might further inhibit the early aggregation at a high concentration.
Due to their high effectiveness in inhibiting Aβ40 and Aβ42 fibrillation, SA and DMyr were also tested for their efficacy in reversing fibrillation. Herein, 25 µM of aged Aβ40 and Aβ42 were incubated at 37 °C alone or with compounds (SA, Gn Rb1, and DMyr) at different molar ratios (Aβ: compounds; 1:5 and 1:10) for 24 h. Assessment of fibril content of the samples at different time intervals was done by the Th-S assay.
Addition of micromolar concentrations of SA or DMyr to the existing fibrils of Aβ40 and Aβ42 led to their disaggregation in a concentration-dependent manner, as determined by a decrease in Th-S signals. This effect was obvious after 3 h of incubation for both Aβ40 and Aβ42 fibrils, with the Th-S signals showing approximately 1/5th of the control samples (Aβ40 and Aβ42 fibrils alone). The control samples without SA or DMyr did not show any decrease in the Th-S signal.
The third tested compound, Gn Rb1, had no significant effect on the disaggregation of Aβ40 or Aβ42 fibrils given the comparable Th-S signals observed with the control sample at different ratios.
It has been reported that the process of aggregation follows a nucleation-dependent pathway [23]. Short fibrils or seeds have been described to speed up the nucleation phase of the aggregation process in vitro and in vivo via a process known as seeding [23]. Hence, we investigated whether the compounds could inhibit the seeded aggregation of Aβ40 and Aβ42. Briefly, Aβ40 and Aβ42 fibrils were fragmented by sonication to short fibrils, which were used as ‘seeds’. These seeds were then added to Aβ40 or Aβ42 monomers and the samples were incubated at 37 °C for 6 h. In the control samples, the additions of seeds accelerated the aggregation of both Aβ40 and Aβ42 monomers, as observed by the increase in the Th-S signal. Interestingly, the addition of SA or DMyr was found to block the seed-induced aggregation of Aβ40 and Aβ42 in a concentration-dependent manner. Moreover, there was > 90% inhibition of seeded aggregation observed when compounds were used at a 1:10 molar excess concentration of SA or DMyr. However, Gn Rb1 had no significant effect on the process of seeded aggregation of both peptides given the comparable Th-S signals with control at different ratios.
