1.0 Molecular Mechanisms of Pathology: Possible Hypotheses

Alzheimer's disease
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Image courtesy of ZME Science

Alzheimer’s disease (AD) is of major concern for the aging population due to its age-dependent onset, and progressive cognitive deterioration of the affected individual[1]. It is known that AD is the primary cause of dementia in the elderly, as well as a leading cause of disability and death[2]. AD is also associated with the significant loss of neurons, and cerebral and hippocampal atrophy[3]. Although the actual process of how the disease begins to arise is not for certain, its development has been suggested to be caused by a succession of events occurring over prolonged periods of time within the brain[1]. Distinct brain lesions further characterize AD, involving neurofibrillary tangles (NFTs), and extracellular plaque formation[4]. The prevailing hypotheses that have been proposed to account for the changes occurring within the brain in AD encompass: oxidative stress, β- amyloid fibril and tau protein hypotheses. These postulations are all interrelated and serve as potential mechanisms for the underlying medical condition.

Effects of Oxidative Stress

Figure 2
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The generation of reactive oxygen species (ROS) by interactions with the copper metal ion.
ROS generation results in various molecular and lipid interactions,
ultimately strengthening the Alzheimer’s pathology. (2013, Zhao, B.).

Functional oxidation reactions occur within the human body repeatedly, however, with excessive oxidation, oxidative stress (OS) is hypothesized to be accountable for the dysfunction and death of neuronal cells. OS is due to the accumulation of reactive oxygen species (ROS), with unstable free radicals that leave membranes and molecules prone to attack[5]. The brain is most vulnerable to free radical damage due to the increased amount of oxygen consumption compared to other body organs, its lipid content, and the scarcity of antioxidant enzymes present[6]. An example of such effects from ROS involves the deregulation of intracellular Ca+ signaling, resulting in the stimulation of apoptotic pathways[5]. ROS are generated by the interaction of molecular oxygen with the redox metals, copper and iron, seen from figure 2 below[5]. Concentrations of these metals are seen to increase in abundance in the brain over time, thus increasing reaction probability within the brain[5]. In addition, evidence demonstrating OS as a probable cause for AD involves the elevation of protein, DNA, and RNA oxidation markers, named 3-nitrotyrosine, 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxynonenal, respectively. Lipid peroxidation products such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and F2-isoprostanes are also elevated within the central nervous system, and localized to neuronal synapses – suggesting an involvement in the loss of synaptic transmission associated with the AD[7]. There is also evidence to suggest that the expression and function of the antioxidant enzyme, superoxide dismutase (SOD), is also affected and related to the progression of ROS effects[7]. The video below helps to elucidate the grand picture created when the hypotheses are joined together.

Amyloid Beta-Induced Neurotoxicity

Figure 3
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The proteolytic cleavage of amyloid precursor protein by alpha, beta, and gamma secretase.
The non-amyloidogenic pathway is catalyzed by alpha secretase; amyloidogenic pathway is catalyzed
by beta secretase; gamma secretase catalyzes the transmembrane region of the protein. (2006, Van Dam, D.)

Extensive ongoing research has been centered upon the notion of Amyloid beta (Aβ) proteins involvement within the pathology of AD. Aβ is generated from the proteolytic cleavage of the amyloid precursor protein (APP) at three distinct sites[8]. This is seen from figure 3 below, demonstrating the three catalase enzymes involved in the process. The beta secretase is also called the beta-site AP cleaving enzyme 1 (BACE1), which is a membrane bound protease. BACE1 cleaves APP at the N-terminus, producing a smaller amino acid fragment, which is then further cleaved by the multi-protein complex gamma secretase within the transmembrane region. This results in the release of a final Aβ protein[2]. Cleavage mediated by alpha secretase results in the toxic form of Aβ proteins[2]. An increase in this proteins production, or a decrease in its clearance eventually causes accumulation associated with plaque formation. Consequently, this is associated with gradual synaptic degradation, neuronal cell damage, and cognitive decline[2].

In terms of oxidative stress, there is a strong implication that there is a causal relationship present regarding Aβ-induced neurotoxicity. It has been postulated by Aksenova et al., 2001, that the precise mechanism of how the principal form of Aβ, (1-42) causes oxidative stress and neurodegradation is not completely understood. Thus, they studied the truncated form of the protein, examining the effects of Aβ(25-35), which mimics the oxidative and physiological effects of the parent protein[9]. Results from this study showed that Aβ(25-35) causes an increased amount of oxidation, and thus is more toxic than the longer Aβ protein. This is suggested to be due to the presence of the methionine residue, positioned at the C-terminus of the truncated protein, which is normally positioned in the middle of the parent protein Aβ(1-42). Modifications to change the C-terminus of Aβ(25-35), such as including the proceeding amino acid valine, to create Aβ(25-36), inhibited the oxidative and neurotoxic effects associated with the peptide. This suggests that methionine plays a critical role in both proteins behavior[9].

Studies performed demonstrated in vitro models administering Aβ treatments that resulted in the increase of hydrogen peroxide and lipid peroxide molecules[2]. Further studies involving how oxidative stress may enhance Aβ production within cells demonstrated that OS dampens the intrinsic activity of the alpha secretase enzyme, while concurrently enhancing the expression and activity of the beta and gamma secretases, to create Aβ(46-50)[2]. Activation of these two enzymes is mediated through the c-Jun-N-terminal kinase (JNK) pathway, which is also actively stimulated by molecules involved in inducing OS[2]. JNK pathway and beta, gamma secretase activation both have been found within the brains of AD patients, thus it possible that increased OS present within AD further enhances the production of Aβ through these mechanisms. In addition, JNK pathway activation has also been implicated in Aβ-induced neuronal apoptosis, thus OS can also mediate Aβ induced neurotoxicity as well as increased Aβ production[2].

Lipid Membrane Interactions

Figure 4
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Mechanistic interactions mediated by the lipid membrane, ROS, and physical aging,
resulting in fibril formation and the induction of Alzheimer’s disease.
(2011, Axelson et al.)

In addition to the effects of Aβ formation, a second hypothesis for AD, involves physical attacks on lipid membranes. This is due to the ability of Aβ proteins to penetrate the lipid bilayer, and while doing so; motivate the membrane to become more permeable to other molecules and cause membrane thinning[8]. Membrane permeability activates phospholipase A2, which promotes membrane fusion[8]. In addition, there is a bidirectionality present between lipid membranes and Aβ proteins, such that lipid membranes can also instill damage to Aβ proteins[8]. Lipid membranes containing PUFA (polyunsaturated fatty acid) chains are also vulnerable to OS. It is suggested that omega 3 fatty acids and antioxidant treatment can aid in reducing the risk of these effects. Aβ has the potential to increase the production of HNE from PUFA chains resulting in His residue modifications in the Aβ protein and increased fibril formation[8]. These interactions function similarly to allow fibril formation. As seen in figure 4, there are many possible mechanisms for the molecular induction of AD, all of which are interconnected and result in the pathogenesis.

ATP-binding cassette (ABC) transporters

The ATP-binding cassette (ABC) transporters have recently been recognized to play a role within the pathology of AD. The prominent transporters involved are the ABCB1, ABCC1, ACBG2, and ABCA1, which transport a variety of lipophilic and amphipathic molecules by ATP hydrolysis[10]. The ABC transporters have been suggested to control levels of Aβ within the brain, such that changes in transporter expression and activity contribute to the Aβ aggregation[10]. Cognitive decline in AD is partially due to impaired protein clearance across the blood brain barrier (BBB) by these transporters, resulting in Aβ accumulation within brain blood vessels, known as cerebral amyloid angiopathy (CAA)[10]. Further modifications in the transporters also influence Aβ production and deposition, which promote amyloid aggregation[10]. Thus, the ABC transporters are ideally distributed in order to maintain cranial homeostasis and control Aβ levels.

Tau Proteins

The pathogenesis of AD is characterized by the presence of extracellular amyloid plaques, as well as intraneuronal neurofibrillary tangles (NFT). The NFTs are composed of hyperphosphorylated Tau protein, which accumulates within the CA1 region of the hippocampus and subiculum, specifically within the entorhinal cortex and pyramidal neurons[10]. Normally, tau is found within axonal membranes[11]. The synthesis of tau aids in creating the polarity of the neuronal cell, since tau contains an acidic N-terminal domain, a basic and proline rich mid section, basic domain and a C-terminal domain[11]. Therefore, the tau protein is hydrophilic and soluble within the membrane, and can be phosphorylated at many cites along its structure[11]. The abnormal phosphorylation of the tau protein interrupts binding with tubulin, and interferes with the promotion of microtubule assembly[2]. This results in the self-aggregation into filaments, causing the NFTs[2]. Molecules associated with the hyperphosphorylation of Tau involve glycogen synthase kinase-3 beta (GSK-3 beta), cyclin-dependent kinase5, mitogen-activated protein kinase (MARK), calcium-calmodulin kinase, and protein kinase C (PKC)[2]. MARK phosphorylates tau at specific KXGS motifs, resulting in the detachment of tau from the microtubules. Thus, tau normally aids in providing stability to microtubules.

Oxidative Stress and Tau

OS is also involved the tau pathology of AD, such that cells overexpressing the tau protein are more susceptible to oxidative damage[2]. This interrelationship between OS and tau proteins was demonstrated in a study involving P301S and P301L tau gene transgenic mice models, with mutations. The results demonstrated that the mice had an accumulation of hyperphosphorylated tau proteins and developed NFTs and neurodegradation[2]. This was in addition to increased OS present within the brains of the transgenic mice, as determined from the OS markers previously mentioned[2]. In addition, Aβ and tau pathological characteristics also involved mitochondrial disruption. This involved the dysfunction of mitochondrial complex IV by Aβ, and the dysfunction of complex 1 by tau proteins[2]. This was determined to be correlated with increasing age, suggesting a decline in mitochondrial respiration processes, decline in ATP synthesis, increased abundance of free radical molecules, which can ultimately lead to synaptic loss and neuronal death[2].

Pathological Modifications of Tau in AD

Figure 5
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Model of Tau mediated microtubule stabilization, demonstrating how the
hyperphosphorylation of Tau results in microtubule destabilization, eventually forming
paired helical filaments, the precursor of neurofibrillary tangles, as seem in Alzheimer’s disease.

Within AD, the intrinsic properties of tau seem to change depending on the severity of the disease[11]. As previously mentioned, in AD, the tau protein becomes hyperphosphorylated at many sites along its structure, which normally occurs before aggregation[11]. This can be seen within figure 5 below. As a consequence, tau also exhibits a decline in microtubule binding, detaching itself from microtubules. This is accountable for the loss of microtubules present within cells and inhibition of intracellular traffic[11]. Tau is also redistributed to a somatodendritic position, in contrast with its native position at the axon. This implies a defect in the trafficking of tau, possibly brought upon by the increased expression of tau mRNA[11]. Tau also becomes aggregated, and becomes what is known as paired helical filaments (PHF) due to their appearance. The PHFs are what cause the NFT to form, due to bindles of PHFs joining together[11]. These modifications in addition to the effects of OS, thus increase the negative physiological effects associated with AD.

Hypotheses Associated with Alzheimer's disease
Summative video demonstrating the events that occur intracellularly
during the onset of disease.
1. Alzheimer’s Disease. (2011). Alzheimer’s disease Education and Referral Center. Publication No. 11-6423.
2. Zhao, B., Zhao, Y. (2013). Oxidative Stress and the Pathogenesis of Alzheimer’s Disease. Oxid Med Cell Longev. 2013: Article ID: 316523.
3. Abuznait, A., Kaddoumi, A. (2012). Role of ABC Transporters in the Pathogenesis of Alzheimer’s Disease. Am Chem S. 3: 820-831.
4. Harman, D. (2006). Alzheimer’s Disease Pathogenesis: Role of Aging. Ann NY Acad Sci. 1067: 454-460.
5. Barnham, K.J., Bush, A, I., Masters, C.L. (2004). Neurodegenerative Diseases and Oxidative Stress. Nat Rev Drug Discov. 3: 205-214.
6. Markesbery, W.R. (1997). Oxidative Stress Hypothesis in Alzheimer’s disease. Free Radical Bio Med. 23: 134-147.
7. Zhao, B., Zhao, Y. (2013). Oxidative Stress and the Pathogenesis of Alzheimer’s disease. Oxid Med Cell Long. 2013: 1-10.
8. Axelson, P.H., Komastu, H., Murray, I. (2011). Oxidative Stress and Cell Membranes in the Pathogenesis of Alzheimer’s disease. Physiol. 26: 54-69.
9. Aksenova, M., Butterfield, D.A., Kansaki, J., Lauderback, C., Varadarajan, S. (2001). Different Mechanisms of Oxidative Stress and Neurotoxicity for Alzheimer’s Aβ(1-42) and Aβ(25-35). J Am Chem Soc. 123: 5625-5631.
10. Abuznait, A.H., Kaddoumi, A. (2012). Role of ABC Transporters in the Pathogenesis of Alzheimer’s disease. Am Chem Soc. 3: 820-831.
11. Mandelkov, E., Mandelkov, E-M. (1998). Tau in Alzheimer’s disease. Trends Cell Biol. 8: 425-427.

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