Alzheimer’s Disease—A Panorama Glimpse

The single-mutation of genes associated with Alzheimer’s disease (AD) increases the production of Aβ peptides. An elevated concentration of Aβ peptides is prone to aggregation into oligomers and further deposition as plaque. Aβ plaques and neurofibrillary tangles are two hallmarks of AD. In this review, we provide a broad overview of the diverses sources that could lead to AD, which include genetic origins, Aβ peptides and tau protein. We shall discuss on tau protein and tau accumulation, which result in neurofibrillary tangles. We detail the mechanisms of Aβ aggregation, fibril formation and its polymorphism. We then show the possible links between Aβ and tau pathology. Furthermore, we summarize the structural data of Aβ and its precursor protein obtained via Nuclear Magnetic Resonance (NMR) or X-ray crystallography. At the end, we go through the C-terminal and N-terminal truncated Aβ variants. We wish to draw reader’s attention to two predominant and toxic Aβ species, namely Aβ4−42/ and pyroglutamate amyloid-beta peptides, which have been neglected for more than a decade and may be crucial in Aβ pathogenesis due to their dominant presence in the AD brain.


Introduction
Alzheimer's disease was first recognized by Alois Alzheimer as presenile dementia in 1906. It is mainly diagnosed in people whose ages are over 65 with the prevalence of Alzheimer's Disease (AD) being shown to grow exponentially with age. It is prevalent among 10% of elderly people, which makes AD an emerging social health issue with the rise of an aging population in the coming decades. Specially, at the age of 85, 50% of the people face the risk of developing AD [1,2]. However, AD is not exactly an aging-related disease [3]. In fact, it has been classified into two types. One is the gene-related heritable AD, known as the early onset familial Alzheimer's disease (fAD). The clinical symptoms can appear in a very young age and it accounts for 25% of all AD cases [4]. Another type is the sporadic Alzheimer's disease (sAD), which constitutes the vast majority of AD cases and is also apparently influenced by genetic contributions besides non-genetic environmental factors [5][6][7]. Several genes have been identified to increase the chance of developing fAD and sAD. However, the pathological role of the only identified 4 lipoprotein E (APOE) gene in sAD is still unclear. In addition, the mutation of APOE 4 is not necessary to increase the risk of developing sAD [8,9]. Thus, the late-onset AD degenerative process has been speculated to be polygenic with the involvement of multiple risk factors [6].
It is well known that the early symptoms of AD include loss of short-term memory, difficulties in executing daily life activities, and withdrawal from social life. The behavioral symptoms include progressive decline in memory, spatial reasoning, attention, and languages. AD is mainly characterized by two pathological hallmarks: the intracellular neurofibrillary tangle (NFT) formed by hyperphosphorylated tau proteins, and the extracellular amyloid plaque consisting of amyloid β peptides. Significant selective neuronal degeneration and loss, with neurotransmitter deficits and inflammations are also evident [10]. In the following section, we first delve into the root of AD by giving a short review on genetic risk factors. After which, we shall discuss on two focal areas of current AD research. One involves the intracellular accumulation of tau protein while the other is on extracellular amyloid aggregation. The former is covered in Section 3 and the latter is addressed in Sections 4-6. In Section 7, we connect these two aspects of AD research to provide a panoramic view of the generative mechanism of AD. We briefly discuss on the progression pathway of AD and the possible therapeutic approaches in Section 8. Finally, we conclude our review in Section 9.

Genetic Revelation of AD
With the higher level of gene expression in the brain, the cumulative DNA damage may have a cascading effect on the transcriptional effectivity and fidelity, and the alteration of DNA conformation in the hippocampus region has been observed in the brain of AD patients [11,12]. For the two types of Alzheimer's-early onset (a.k.s. familial AD) and later onset (a.k.s. sporadic AD), both have a genetic connection. Familial AD involves a number of single-gene mutations on chromosomes 1, 14 and 21, which corresponds to the abnormal presenilin 2, presenilin 1 and amyloid precursor protein production respectively [13][14][15][16][17], and each of these mutations is believed to play a very important role in the cleavage of APP and thus affect Aβ production. The late-onset AD accounts for the major cases of AD, notwithstanding a lack of full understanding, the genetic risk factors, such as the definitively identified apolipoprotein E (APOE) gene on chromosome 19 [18] and the methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) gene on chromosome 6 [19] as well as some other loci [20][21][22][23], are likely to affect the predisposition of AD. Additionally, the genome-wide study of AD cases also revealed new novel variants, which may modify the age of the AD onset or show gender-linked susceptibility [24,25].

Tau Protein and Tau Accumulation
Tau protein arises from the alternative splicing of the microtubule-associated protein tau (MAPT) gene, and is abundant within the central and peripheral nervous systems. It is one of the intrinsically unstructured proteins (IUPs), which exhibit as random coils under physiological conditions, and are capable of folding into well-defined stable structures, e.g., the neurofibrillary tangles in AD. Normally, it is the phosphorylation-modified tau protein that stabilizes the axonal microtubules in the central nervous system (CNS). Moreover, tau protein with actin cytoskeleton and plasma membrane serve as enzyme anchors, and they are also believed to help in the neurite outgrowth and the transport of axoplasm. However, under certain circumstances, tau protein may undergo abnormal phoshorylation, hyperphosphorylation and some other modifications -nitration, ubiquitination, truncation, shift, prolyl isomerization, which may reduce the binding affinity of tau towards microtube, and thus lead to either intraneuronal accumulation of tau protein or its binding to other macromolecules [26][27][28][29].
The mislocalization and accumulation of tau proteins in dendrites and dendritic spines brings about a disruption of neuronal cell communication, which precedes neurodegeneration and causes a loss of memory [30]. Tau self-assemblies of tau proteins forming straight filaments (SFs) and/or paired helical filaments (PHF) may further aggregate into NFT, which is significantly correlated with the severity of AD. Electron microscopy of PHFs showed the appearance of two strands twisting around each other with a cross-over repeat of 75-80 nm and a width of 10-22 nm [31,32]. It has also been further revealed that β-sheet is the most dominant structure in the PHFs.

Amyloid Precursor Protein
The amyloid β plaques results from the aggregation of the amyloid β (Aβ) peptides, which is cleaved by the βand γ-secreastase from the amyloid precursor protein (APP). The precise biological function of APP is as yet not well defined even though lots of studies have revealed its biological and physiological importance in the neurite outgrowth modulation [33], copper homeostasis regulation [34], synaptic transmission and formation, and synaptic function and activity [35,36]. On the other hand, it has been shown that the absence of APP in a mouse model did not cause a significant impairment of cognitive abilities, but instead led to a decrease in locomotion activity [37].

Aβ Aggregation Pathway
Neuronal impairment is observed in patients even before Aβ plaque formation during the early onset AD. It is generally believed that Aβ oligomer is the main culprit of neurotoxicity [38][39][40][41]. Due to these oligomers being easily attached to the membrane or other macromolecules and hard to be isolated from these structures, conventional experimental studies on them are very difficult [42]. A progress has been made recently which shows that Aβ dimers can be measured and strongly associated with dementia [43]. Besides the experimental methods, molecular dynamic simulation is a complementary approach for atomic-level studies of the unstructured monomer aggregation process, structural evolution and toxicity.

Structural Evolution during Aggregation
In recent decades, Nuclear Magnetic Resonance (NMR) and X-ray have been carried out to determine the structures of Aβ in water and membrane mimic environment (see Table 1). One general observation is that Aβ peptides exhibit great polymorphism. In many situations, Aβ is found to easily attach to other protein, or self-assemble into large oligomers or fibrils. These complex involving Aβ peptides, such as APP, Aβ binding copper ions, Aβ segments with enzymes as observed in experiment, are cataloged in Table 2.  The central amyloidogenic step of the oligomerization process is the transition from α-helix rich (starting from the conformation of APP before being cleavaged) to β-sheet rich structures. All atom simulation of Aβ 37−42 reveals the polymorphism of Aβ oligomers [101]. Replica exchange molecular dynamics simulations were conducted to study the short peptide Aβ 10−35 [102] and Aβ 16−22 [103] dimer and trimer formations as well as the Aβ 16−35 monomer and dimer structure and thermodynamics properties [104]. A coarse-grained model of Aβ 1−42 was used to study the structural diversity of the dimer [105] in aqueous environment. A single Aβ 40 peptide was used to study its structural diversity [106] and the relevant effects of insertion depth and ionic strength in the DPPC membrane environment [107]. Further study shows that the thermodynamics and dynamics of Aβ oligomerization are sequence dependent [108].
Aβ has two alloforms: one is Aβ 1−40 , the other is Aβ 1−42 . Both have distinct effects and pathways during oligomerization [109,110]. It is generally believed that Aβ 1−40 peptides are non-amyloidogentic while Aβ 1−42 are amyloidogentic. Recent studies have shown that Aβ peptides produced in the area with elevated level of cholesterol pose a great risk of Alzheimer's disease [111] and those genes associated with the cholesterol regulation play a significant role in the predisposition of AD. Here, it raises the question on the effect of cholesterol binding to Aβ peptide and the associated mechanisms of Aβ aggregation in the membrane environment. Recent studies done by our group show that cholesterol molecules compete with the intra-action of Aβ oligomers by binding directly with Aβ peptides. This implies that monomeric Aβ and/or small Aβ aggregations prefer to locate within cholesterol-rich membranes [111][112][113]. Furthermore, Aβ structure evolution in the presence of small and macro-molecules, such as curcumin [114,115], heme [116], resveratrol [115,117], mitoxantrone and pixantrone [118], derivatives of Congo Red [119], 1,4-naphthoquinon-2-yl-L-tryptophan inhibitor [120], EGCG [115,121], NqTrp [115], and inflammation protein complex [122], are also studied. In these studies, Aβ aggregation behaviour is found to be either inhibited or promoted.
The single mutation of Aβ are also performed to study the mutation effect on Aβ oligomerization process. A2V mutation in Aβ 1−28 shows that the intrinsic disorder are reduced with a completely different free energy landscape [123]. D7N mutation on the Aβ 40 and Aβ 42 exhibits a notable change in secondary structure, final topology and salt bridge compared with wild type [124]. D23N mutation also causes a distinct dimerization pathways compared with wild type in Aβ 1−42 and Aβ 1−40 [110].
The detailed fibril nucleation and oligomerization are further probed from kinetics and thermodynamics aspect via computational method [125].

Amyloid Fibril Formation and Polymorphism
Since the end of the 20th century, much effort has been made to understand the structure of amyloid fibrils and the mechanism of its formation. With the development of measurement techniques, such as NMR and X-ray crystallography, the polymorphic structures of amyloid fibril have been revealed. The main contribution to the distinct structures of the fibril is the sensitivity of the fibril growth towards the surrounding conditions [126][127][128]. Despite the multiple differences in their overall structures, there exists a common a well-characterized antiparallel β-sheets within the fibrils [127]. Molecular dynamics simulations have been employed to investigate the detailed mechanisms of the Aβ fibril formation by adding monomers into the structured oligomers [129]. It was found that the incorporation of the monomers into the oligomers occurs in two distinct stages: the first stage is a rapid conformational change of the monomers from a disordered structure to one with a significant amount of beta-strand content. The second stage is a relatively slow process, namely the docking of the monomer which has adjusted itself into a well-registered antiparallel structure.

Aβ Isoforms -Variants of Aβ
The amyloid-cascade hypothesis holds a stronghold in the research of AD. It posits that the process of Aβ aggregation into oligomers and final deposition as plaques is the central pathological events in AD. As stated earlier, Aβ 40 and Aβ 42 are two well-recognized isoforms of Aβ being produced. Meanwhile, with the conduct of intensive research in this area, several C-terminal truncated isoforms, such as Aβ 43 [130,131], Aβ 1−15/16 [132], and carboxyterminally truncated Aβ peptides 1 − 37/38/39 [133], have been revealed and they are suggested to play a crucial role in the AD pathogenesis. In particular, experimental data obtained from both sporadic and familial AD shows that Aβ 43 is more prevalent than Aβ 40 in plaque core [130].

Aβ Oligomers and Tau Protein: Relationship and Link
There are various means by which Aβ oligomers are distributed among cells. These are: diffusion or spread within extracellular parenchymas as oligomers or deposited plaques; adsorption on membrane surface or incorporation into membrane structure forming pores or channels; and accumulation within the neuronal structure. On the other hand, the tau protein is mainly distributed within the intracellular neuron. One possible direct link between the intraneuronal Aβ and tau protein involves the modulation effect between Aβ and tau pathologies [146].
Studies have shown that intracellular accumulation appears earlier than amyloid plaque and NFT, and have suggested that intraneuronal Aβ accumulation initiates the caspase-cleavage of tau and precedes the Aβ plaque and NFT formation [147,148]. Meanwhile, tau hyperphosphorylation signal transduction pathways may also be linked indirectly to Aβ oligomers. Recent reviews on the relationship between Aβ pathway and tau pathology can be accessed from references [146,149].

AD Progression Pathway and Current Therapeutic Strategies
Mild cognitive impairment (MCI) has been used to prescribe the transitional stage between healthy brain and dementia. One impairment subtype is amnesic mild cognitive impairment (aMCI), which may increase the risk of progression to AD. Due to a variation in definitions of MCI based on different clinical criteria, the pathology of aMCI still lacks a strong characteristic profile. In terms of the intermediate stages towards AD, MCI shares a lot of similarity with AD, i.e., an increase of NFT in the medial temporal lobe (namely, hippocampus) amygdala.
In the last few years, donepezil, rivastigmine, and galantamine are prescribed drugs for AD patients to target acetylcholinesterase that inhibits the breaking down of acetylcholine. Another drug memantine has been used to block glutamate receptors against excitotoxicity as a means to cure AD. To date, the acetylcholinesterase inhibitors are the most widely used AD drug and have been to some extent successful in slowing down the process of cognitive impairment [150].

Conclusions
Alzheimer's disease is a complex and progressive neuro-degenerative disease. There are numerous studies from different points of view on the pathology of AD, such as those mentioned in this review, which involve genetic and environmental factors, tau protein and neurofibrillary tangles, the variety of its isoforms as well as amyloid beta peptides and oligomers. However, all of these issues are not isolated. In all likelihood, the actions among extracellular amyloid β peptides and intracellular tau proteins are closely related to each other through a series of complicated, but essentially important, processes and events. Despite the strong links between Aβ and tau protein that have been reported so far, a panorama study of these deeply connected roadmap is still missing. In order to explore the whole landscape of AD, a step by step strategy is of paramount importance, such as the uncovering of the mechanism of Aβ peptide aggregation, which will help to decipher the whole story on the pathogenesis of AD.