Current study on diagnosis and treatment of Alzheimer’s disease by targeting amyloid b-protein

Introduction

Alzheimer’s disease (AD) is one of the most common degenerative diseases of the brain. It is caused by multiple genetic and environmental factors. AD occurs more frequently in the elderly over 65 years old. The main manifestation of AD is dementia. The course of AD varies, the average duration is seven years, and most people die of complications. In addition to the age of onset, the disease is also characterized by familial aggregation, resulting from the interaction between genetic and environmental factors [16]. In a 2018 survey, more than 10 million people in China suffered from AD, almost doubling every ten years [61]. Over the past 100 years, many pathophysiological aspects of AD have been discovered and identified. Many clinical studies have shown that the onset of AD may be induced by a variety of factors, including excessive aggregation of amyloid b (Ab) and neurofibrillary tangles (NFTs) formed by hyperphosphorylation of tau protein, absence of the neurotransmitter acetylcholine (ACh), activation of microglia by inflammatory reactions, and brain cell damage caused by advanced glycation end products (AGEs) with their receptors (RAGE) [45,40], and so on. Also, researchers found that there were significant gender differences in the incidence of AD after the age of 85 years. At the age of 90, the incidence of AD is three times higher in women than in men [2]. There is a significant interaction between sex and Ab42 on longitudinal hippocampal atrophy, and longitudinal decline in memory and executive function [28]. A recent study shows that follicle-stimulating hormone (FSH), whose serum level is rising sharply in women before the last menstrual cycle, acts directly on hippocampal and cortical neurons to accelerate Ab and Tau deposition and impairs cognition in mice displaying features of AD [66]. FSH can phosphorylate AKT, ERK1/2, and SRPK2, leading to the activation of C/EBPb-AEP/d-secretase and the cleavage of APP [66]. The study suggests that a highly targeted anti-FSH antibody could be a new way to treat AD [66]. Currently, AD can be alleviated by drugs but cannot be cured. With the aging issue in China becoming increasingly severe and the number of AD patients increasing year by year, it is essential to explore the treatment of AD [65]. We here review the current research progress in the treatment of AD by targeting Ab and hope to provide some references for developing new drugs for AD.

The formation mechanism and types of amyloid b

As early as last century, scientists had discovered the presence of Ab fibrillary protein in the brain of AD patients by electron microscope observation and po-inted out that Ab was related to the occurrence of AD [26]. Ab is a kind of polypeptide highly resistant to proteolysis and is produced when amyloid precursor protein (APP) is cleaved by corresponding proteases. Two pathways are responsible for APP cleavage, one of which is the usual pathway and the other Ab cleavage pathway (Fig. 1). Ab is composed of 37-43 amino acids, of which Ab40 and Ab42 are the most common subtypes [10]. Most Ab peptides have a length of 40 residues (Ab40), while a small number have a length of 42 residues (Ab42). Ab42 is generally considered more neurotoxic due to the increased possibility of misfolding and protein aggregation caused by the binding of two residues [1]. In addition, the low solubility of Ab42 makes it more likely to accumulate and form fibrous substances, which then form Ab plaques. Ab protein can accumulate in the extracellular matrix after releasing from cells by exocytosis [4,60]. The lysosome can degrade the Ab protein. However, as people get older, the permeability of the lysosome membrane changes, making Ab protein outflow more easily to result in the accumulation of Ab protein. Meanwhile, the lysozyme activity also decreases, leading to a decrease in dissolving efficiency and the accumulation of Ab protein. The accumulation of Ab protein leads to an increase in Golgi vesicles, causing dendrites to swell and shorten and impeding neurotransmitter transmission [38,41].

Neuronal effects of amyloid b on neurons at different levels

N-methyl-d-aspartate receptor (NMDAR) mediates transmission and plasticity changes between excitatory synapses. The subunits discovered so far are NR1, NR2 (A-D), and NR3 (A, B) [19]. Under normal circumstances, Ab promotes the release of glutamate from vesicles into the synaptic cleft and acts on the NMDAR on the postsynaptic membrane. When excess glutamate is released and astrocytes are dysfunctional, glutamate can accumulate in the synaptic cleft and cause the activation of NMDAR outside the synapse, leading to calcium overload in postsynaptic neurons and inducing excitatory toxicity, neuronal apoptosis, and neurodegeneration [29].
In previous studies, researchers investigated the ef-fects of Ab42 on neuronal excitability and voltage-gated sodium channels using a cellular patch-clamp, and they found that Ab42 can reduce the threshold of neuronal action potential and increase the firing frequency of neuronal action potential [17]. Inhibition of sodium channels and excitability may be a target for treating AD.
In addition, exogenous Ab40 may reduce the survival rate of neural stem cells and reduce the proportion of neural stem cells that differentiate into neurons by inducing neural stem cell apoptosis. This can lead to a decrease in the number of neurons within the human brain and ultimately to the onset of AD [5].
Many studies in animal models and ex vivo cultures indicate that amyloid b-peptide oligomers induce synaptic damage early during the progression of AD [11]. And further research showed that the Ab oligomers exhibit early dual effects by acutely enhancing dendritic complexity and spine density [50].

Amyloid b-based diagnostic method

In 2007, the International Working Group (IWG) launched a new study on AD diagnosis, presenting a conceptual framework with criteria introducing biomarkers into the diagnosis of AD [12]. With the development of non-invasive diagnostic techniques, a new method for improving diagnostic accuracy has emerged. Positron emission computed tomography was used to detect the deposition of Ab in living brain plaques after injection of radiolabelled tracers [8]. For example, in recent years, several fluoro-18 (18F)-labelled amyloid tracers have been developed [68]. 18F-florbetaben Ab imaging facilitates accurate detection of prodromal AD. In addition, Ab detection in cerebrospinal fluid (CSF) can also assist in AD diagnosis, but the accuracy is slightly lower than that of 18F-florbetaben Ab imaging [20]. Early this year, researchers used a functional probe responding to Ab oligomer via second near-infrared window (NIR-II) fluorescence imaging, which is more sensitive to Ab oligomer (Kd = 6.16 nM) than Ab fibrils. In addition, this method showed no response to other small biological molecules, proving to be conducive to the early diagnosis of AD in vivo [33]. In addition, because Ab can migrate from the blood-brain barrier to plasma, studies have assessed Ab40 and Ab42 levels in human red blood cells (RBC). With evidence of an age-dependent increase of Ab concentration in RBCs, confirmation of RBC-associated Ab may be a tool for early recognition of dementia and predicting cognitive impairment [32].

Treatment of Alzheimer’s disease with amyloid b protein as a therapeutic target

Inhibition of Ab generation

Multiple studies have shown that Ab oligomers and plaques at all stages of AD can produce direct neurotoxic effects. It has been recently proposed that in the predementia stage of AD, removal of the negative impact of Ab pathology may improve memory and hippocampal function [13]. Therefore, reducing the production of Ab polypeptide to minimize the formation of Ab oligomer and plaque expansion is of great significance for the treatment of AD [62].

Activation of a-secretase

A disintegrin and metalloproteinase (ADAM) is a family consisting of transmembrane proteins and secretive metalloproteinases composed of approximately 750 amino acids. It functions by promoting cell adhesion and proteolysis [23]. ADAM10 is a major a-secretase that inhibits Ab peptide formation from APP in the non-amyloid pathway. Enhanced ADAM10 activation can promote the generation of soluble APP-a (sAPPa), a derivative fragment of APP, thus protecting the brain by inhibiting Ab production [67]. This fragment has been shown to exert neuroprotective effects [36]. Since both Ab and sAPPa are sequentially proteolysed products of APP protein, its production can be reduced by increasing the generation of sAPPa [1,10]. It was found that a-secretase can not only increase benign APP metabolism but also inhibit the activity of g-secretase in the HEK293 cell line [56].
Since a-secretase has few side effects, enhancing its activity is a potentially effective treatment for AD. Previous studies have demonstrated that Acitretin can promote the increase of mature ADAM10, thus increasing the production of a benign metabolite of APP [14,52]. Since APP processing can regulate lipid metabolism, lipid homeostasis should be closely monitored during the application of Acitretin to avoid hyperlipidaemia [31]. Etazolate, a kind of neuroprotective drug linking GABA(A), was shown to activate a-secretase and increase sAPPa production [35] (Fig. 1).

Inhibition of b-secretase

b-secretase (BACE) is a transmembrane aspartate protease that cleaves APP to generate sAPPb and its C-terminal fragment CTF-b. BACE is the main target enzyme for treating AD [51].
b-secretase-1 (BACE-1) expression levels are increased in most patients with AD, and APP cleavage by b-secretase can produce pathological products [59]. Therefore, BACE may be a significant therapeutic target for AD. Over the years, b-secretase inhibitors, such as LY2811376 and LY2886721, have often been discontinued due to toxic side effects [39]. Researchers identified TA-III in Rhizoma Anemarrhenae (RA) as a novel BACE-1 inhibitor by combining cell extraction and chemical genomics target guidance, and verified its potential pharmacological target as BACE-1 using surface plasmon resonance (SPR), enzyme activity measurement, and visual molecular docking method. TA-III was confirmed to prevent neuronal injury by improving memory impairment, reducing Ab aggregation through the amygdala pathway, and impairing the NMDAR/ERK signalling pathway [63] (Fig. 1).

Inhibition of g-secretase

g-secretase is a kind of intramembrane aspartic acid protease, a member of the intramembrane-cleaving-proteases (I-Clips). g-secretase is comprised of presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) [27]. b-secretase cleaves APP to produce two fragments, while the final step of Ab production requires g-secretase to cleave the CTF-b fragment. Because of its important role in Ab production, g-secretase has also become an important drug target. Blocking g-secretase cleavage of APP can reduce the amount of toxic Ab and ultimately achieve the purpose of treating or alleviating the progression of AD [9]. Begacestat, a novel, 2,5-disubstituted thiophene sulfonamide from Wyeth (now Pfizer), has been identified as a potential g-secretase inhibitor [37]. It has also shown promise in clinical trials [22]. In 2016, researchers reported the characterization of FRM-36143, a compound derived from optimizing a novel structural class of g-secretase modulators (GSMs). It has been confirmed to reverse the effect of PS mutations, which may benefit the patients when treated early enough in the disease [6] (Fig. 1).

Drug that antagonizes amyloid b

Of the four targeted drugs with potential for approval [57], Alz-801 is an optimized precursor drug modified from taurine, a kind of Aβ antagonist. The metabolic product of tramiprosate and the precursor drug ALZ-801 in humans is 3-sulfopropionic acid (3-SPA), whose level is 12.6 times higher in AD patients treated with tramiprosate than in untreated patients. Multiple 3-SPA molecules can interact with the Ab42 molecule to inhibit its polymerization into oligomers, significantly affecting AD patients with the apolipoprotein E (ApoE) e4 gene.

Promoting the clearance of amyloid b

Transportation through the blood-brain barrier
The deposition of Ab may begin years or even more than ten years before the onset of AD, so early intervention to remove Ab is crucial [48]. Extracellular deposits of Ab can be removed from the brain by various clearance systems, most notably by transportation through the blood-brain barrier (BBB) [54]. Earlier studies in mice showed that about 75% of total extracellular Ab can be cleared through the BBB, and only a few can be cleared by interstitial fluid (ISF) [43]. Subsequently, researchers found that CSF enters the parenchyma along paravascular spaces surrounding penetrating arteries and that brain interstitial fluid is cleared along paravenous drainage pathways. Loss of the Aquaporin 4 (Aqp4) gene inhibits the clearance of soluble Ab during CSF mass flow, suggesting that this pathway may play a role in the removal of Ab from the central nervous system [24].

Amyloid b clearance by microglia and astrocytes

Studies have shown that Ab aggregation induces the activation of microglia. Although activated microglia have the role of phagocytosis and clearance of Ab to reduce the damage of Ab to brain tissues, they can secrete a large number of cytotoxic substances, thus aggravating the inflammatory response [21]. Ab induced reduction of lysosomes possibly through inhibition of mTOR/TFEB signalling pathway in both neurons and microglia in AD transgenic mice. The promotion of lysosome degradation can effectively reduce immature autophagosome accumulation, accelerating autophagosome clearance of Ab and other pathological proteins [53]. This study also offers new ideas for treating AD.
Recent reports demonstrated that low-density lipoprotein receptor-related protein-1 (LRP1) plays a dominant role in clearing excessive Ab production in an astrocyte-dependent clearance pathway [58]. LRP1 regulates the uptake of Ab and degradation in astrocytes, and loss of low-density LRP in astrocytes can reduce the clearance of Ab [34].

Immunotherapy targeting amyloid b

The results using animal AD models suggested that active and passive immunotherapy targeting Ab is promising [64]. However, many side effects have been reported about the active immunotherapy for AD, such as encephalitis and cerebral haemorrhage [42]. AN-1792 was the first active immunotherapy strategy for AD. However, it was interrupted by meningoencephalitis in 6% of immunized patients in a Phase IIa immunotherapy trial [18]. In 2006, researchers constructed an AD mouse model in which Ab derivative was co-administered with alum adjuvant to promote humoral immunity. This immunotherapy can reduce Ab load without increasing vascular Ab deposits or microhaemorrhages at 11-24 months post-treatment [3]. This study also suggests that the Ab derivative, when used with an adjuvant suitable for humans, can reduce Ab load and promote cognitive improvement without increasing vascular microbleeds compared to other Ab antibody studies [3]. In 2021, Fang et al. [15] reported a novel recombinant chimeric 12×(Ab1-15-Th) antigen targeting the pathological confirmation of Ab oligomer for AD treatment. They tested the immunogenicity of the chimeric vaccine in C57/BL6 mice and demonstrated its efficacy in AD mice. The results showed that the chimeric vaccine showed a noticeable neuroprotective effect by reducing the levels of soluble Ab oligomers and soluble Ab in the brain, suggesting that the chimeric vaccine is a good candidate for AD prevention [15]. ABvac40 is an active vaccine against the C-terminal end of Ab40, and an ongoing Phase II clinical trial is needed to explore the clinical efficacy of ABvac40 [30]. In an active immunotherapy study, Ab-targeted antibodies prevent the fibrosis of Ab peptides, destroy preformed fibres, and interfere with the aggregation of Ab [47].
Passive immunization is defined as a prefabricated antibody injection to provide immunity to the host. Examples include Aducanumab, a molecule screened from the human memory B cell family that binds specifically to Ab aggregates, and Gantenerumab, an experimental immunoglobulin G1 (IgG1) antibody that binds specifically to Ab [7]. Both drugs have the role of activating immune response and removing antigen-antibody complexes [46]. In addition, BAN2401 is a humanized IgG1 monoclonal antibody that selectively binds to soluble Ab aggregated species. Previous studies have shown that BAN2401 can reduce amyloid loading on PET examination, slowing cognitive decline in a Phase II study in early symptomatic AD. It is currently tested in Phase III clinical trials to treat AD [44].
In addition to monotherapy, Ab deposition can also be eliminated through passive Ab immunotherapy and b secretase inhibitors. By combining an antibody treatment with chronic BACE1 inhibitor treatment, researchers demonstrate significant clearance of pre-existing amyloid deposits in Balb/c transgenic mice brains without inducing microhaemorrhages and other histopathological findings [25]. Overcoming the BBB is vital for most therapeutic drugs entering brain tissues. Nanoparticle (NP) mediated drug delivery systems prove to be a potential means of enhancing brain tissue-targeted drug transport through BBB [55]. Since nanoparticles can be used as drug delivery carriers across the BBB, the combination of nanoparticles and antibody fragments has been used to diagnose AD [49].

Conclusions

The pathogenesis of AD may be caused by multiple pathways and aetiology. Although there are side effects or insignificant effects of drugs in Phase I and Phase II clinical trials, new targets of drug action have been constantly discovered with the development of scientific research and technology and the deepening of related studies on the pathological mechanism of AD, and relevant drugs have been approved. There are still many candidate proteins targeting Ab under investigation, which brings hope to cure AD. This review is expected to provide some references for developing treatments against AD by targeting Ab.

Acknowledgements

This research was funded by Jiangsu Province Shuangchuang Talent Plan (Grant No. JSSCRC 2021533), College Students’ Innovation and Entrepreneurship Training Program of Jiangsu Province (Grant No. 2021-10295144Y), the Research Start-up Fund of Jiangnan University (Grant No. 1285081903200020), and Research Start-up Fund of Wuxi School of Medicine, Jiangnan University (Grant No. 1286010242190060).

Disclosure

The authors report no conflict of interest.

References