Li, Tian-Tian
(2024)
PHARMACOLOGICAL EVALUATION OF NOVEL COMPOUNDS AS ALZHEIMER’S DISEASE TREATMENT.
PhD thesis, University of Nottingham.
Abstract
Background: Alzheimer’s Disease (AD) is an age-related and progressive neurogenerative disorder characterised by a progressive loss of memory and cognitive impairments. Excessive Aβ deposition and tau hyperphosphorylation are pathological characteristics of AD, which are accompanied by oxidative stress, metal dysregulation, and neuroinflammatory processes. Currently, most medications being tested in clinical trials for AD are anti-amyloid therapies. Nevertheless, given the ineffectiveness of small-molecule medicines targeting Aβ, there has been a shift in the emphasis of drug research towards the creation of medications that target alternative pathways, such as anti-tau, anti-inflammatory, and multi-target therapies. The development of multi-target medications has the benefit of compensating for the inadequacy of modulating a single target to achieve the desired disease-modifying effectiveness in complex, multifactorial disorders like AD. Additionally, neuroinflammation, which is central to AD's pathology, results in synaptic and neuronal damage. Cannabis-derived cannabidiol (CBD) has been shown to be a promising AD drug because it has anti-inflammatory, antioxidant, and neuroprotective properties. CBD has been shown to inhibit the formation and aggregation of tau fibrils and protect against microglial and Aβ-induced neurotoxicity.
Aims: We would like to investigate the therapeutic potency of newly synthesised dual-target inhibitors (19n: AChE + metal ion; C10: AChE + MAO-B) against AD. The test compounds 19n and C10, both with AChE inhibitory activity, and the moiety with another novel ability, could improve efficacy and lead to better outcomes in most multitarget therapeutic design efforts for AD. Therefore, it is interesting to determine whether these two dual-target inhibitors are better than single-target inhibitor (donepezil). AFM has been used in recent years to explore the mechanisms underlying β-amyloid fibrillization, so we would like to further visualize how these two dual-target inhibitors affect Aβ1-42 aggregation induced by AChE. Although CBD is a single drug, it has been shown to affect different drug targets in the brain. We would like to investigate the therapeutic efficacy of CBD in AD and its antiinflammatory effects involving the cGAS/STING and NLRP3-dependent pathways.
Method: Experimental models are essential for further understanding of AD pathology. We utilized chemical/drug-induced models for AD in our projects. Firstly, it has been demonstrated that the okadaic acid (OA)-induced model for AD is effective at screening AD medications. This model can replicate all the pathological hallmarks of AD. We used OA to induce oxidative stress and mitochondrial dysfunction in SH-SY5Y cells. SH-SY5Y cells are similar to cholinergic neurons, indicating that these cells can model the cholinergic system. Therefore, this cell is beneficial for evaluating drugs targeting cholinergic neurons. Based on a previous experiment by our research group, a novel AD model in which glyceraldehyde (GA) is used in SH-SY5Y cells to trigger tau phosphorylation has been developed. In addition to assessing the effects of drugs on tau pathogenesis, this model can also establish the correlation between AD and diabetes mellitus in order to investigate if drugs affect tau by modulating glucose metabolism. Therefore, the GA/OA-induced AD model against SHSY5Y cells is used to investigate the therapeutic potency of newly synthesised dual-target inhibitors in different pathways.
Although the AD cellular model we established before can express various AD pathological features, the role of neuroinflammation in AD is lacking, so we used BV-2 microglial cells treated with 2’3’-cGAMP or FeSO4 as two inflammation models, both of which can activate the cGAS/STING and NLRP3-dependent pathways according to two protocols. Although current data indicate that CBD inhibits NLRP3 inflammasome activation, there is no report on the effect of CBD on the cGAS/STING pathway. The overstimulation of the cGAS/STING pathway and its associated neuroinflammation is believed to contribute to the development of AD. Additionally, a study shows that 2’3’ cGAMP synthesised by cGAS can activate the NLRP3-dependent pathway. Therefore, we would like to explore the anti-inflammatory effect of CBD on the cGAS/STING pathway and the NLRP3 pathway activated by 2’3’-cGAMP.
Moreover, we performed animal studies with a scopolamine/Aβ42-induced AD model in mice. The scopolamine-induced model is a widely accepted model for studying cholinergic deficits. The Aβ42-induced model replicates many pathological hallmarks of AD, which is a specific model for screening AD drugs. AFM and Transmission electron microscopes (TEM) were applied to investigate the inhibitory effect of dual-target inhibitors on Aβ pathology.
Results: We showed that both AChE/metal ion dual inhibitor 19n and AChE/MAO-B dual inhibitor C10 have neuroprotective and antioxidant properties in established cellular models of AD. The decrease in ROS level and increase in mitochondrial membrane potential (MMP) in the OA-induced SH-SY5Y cells indicate that 19n and C10 may prevent ROS damage by inhibiting the p38 MAPK and JNK pathways that are activated by OA. We also assessed the levels of tau abnormal phosphorylation at residues 396 and 199 in the GA-induced SH-SY5Y cells through ELISA to determine the effects of 19n and C10 on tau pathology. We observed that 19n and C10 markedly attenuated GA-induced hyperphosphorylation of tau protein, indicating that they play a role in inhibiting GSK-3, a key enzyme involved in regulating the phosphorylation of tau proteins and glycogen metabolism. Furthermore, we demonstrated that 19n and C10 can inhibit AChE-induced amyloid aggregation. Although 19n and C10 don’t display a higher AChE inhibitory activity than donepezil in the AChE inhibition assay in the SH-SY5Y cells, they are able to reduce Aβ aggregation via AChE inhibition. AFM was used as an imaging technique to measure the aggregation process in the absence or presence of test compounds. Results suggest that donepezil has the strongest inhibitory effect on the length of fibrils. However, the compounds 19n and C10 demonstrated significant inhibitory effects in terms of fibril height; in particular, compound C10 exhibits the highest potency. Compounds 19n and C10 have also reduced the amount of Aβ that forms. TEM findings show that compound 19n can inhibit Cu2+- induced Aβ1-42 aggregation due to its metal chelating properties.
Our animal study showed that compounds 19n and C10 rescue cognitive dysfunction induced by scopolamine in mice, and C10 has better effectiveness than donepezil. This also indicates that C10 may be more effective than donepezil in treating cholinergic deficiency in the early stages of the disease. Compound 19n showed a better ability to penetrate the BBB and better oral bioavailability than compound C10, so we further investigated the effects of compound 19n on oligomerized Aβ1-42-induced cognitive impairment and proinflammatory cytokine production in mice. Our results showed that the compound 19n can ameliorate oligomerized Aβ1-42-induced damage, but it still has not shown better efficacy than donepezil. Additionally, compared to donepezil, 19n has a higher LD50 value, indicating that 19n has a larger safety window than donepezil.
The established inflammation model demonstrates that 2'3'-cGAMP and FeSO4 activate the cGAS/STING pathway in distinct ways. FeSO4 stimulation can induce cGAS to synthesise 2'3'-cGAMP, which subsequently stimulates STING. However, the pathway is initiated by 2'3'-cGAMP, which binds directly to STING and induces upregulation of downstream targets. More cytokines and the IFNβ gene were expressed by BV-2 cells stimulated with 2'3'-cGAMP, as opposed to stimulation with FeSO4. Therefore, our study showed that CBD may have anti-inflammatory effects on 2'3'-cGAMP or FeSO4-induced microglia cells. It reduces the expression of cGAS, STING, and various inflammatory cytokines such as IL-1β, TNFα, and IFNβ in response to the activated cGAS/STING pathway. Our study also showed that CBD has neuroprotective properties regardless of Aβ-induced neurotoxicity or amyloid selfaggregation, as well as tau phosphorylation caused by GA.
Conclusion: Compounds 19n and C10 are involved in many pathologies of AD, such as oxidative stress, tau phosphorylation, and amyloid aggregation, so they are potential lead compounds for AD treatment. CBD may be a promising candidate for the development of novel therapeutic strategies for the treatment of AD and cGAS/STING-associated neuroinflammatory disorders.
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