Cellular Mechanisms in Alzheimer’s Disease E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Recent Advances in Alzheimer Research
- Sprache: Englisch
Alzheimer’s Disease AD is the product of the slow and progressive degenerative alteration that develops in the adult brain and can remain asymptomatic for a considerable time before cognitive deficits becomes evident. The main challenge for researchers
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PREFACE
Alzheimer's disease (AD) is the most common cause of dementia worldwide, several groups are involved in studying the predisposition and disease development sought to understand and treat all its consequences.
AD is the product of the slow and progressive degenerative alteration that develops in the adult brain and can remain asymptomatic for a considerable time before the cognitive deficit becomes evident. The main challenge is to identify markers of this degenerative process, in this sense, data have been generated in the field bringing new mechanisms and hypothesis to explain its pathophysiology. This book intends to review the most recent studies in AD and get new perspectives to explain some aspects of this neurodegenerative disease.
The focus of this book is to discuss the cellular mechanisms in AD including well known theories and new perspectives in the main neuronal signaling pathophysiology (β-amyloid and phosphorylate tau). Current concepts on AD will be presented, bringing the more actual information about the cellular mechanisms and new views to explain the disease progress.
FOREWORD
In 2015, according to statistics compiled by the Global Voice on Dementia produced by the Alzheimer’s Disease International group, there were an estimated 46.8 million people worldwide living with dementia. Recent estimates for 2017 put this number closer to 50 million people. It is projected that this number will double every 20 years attaining 131.5 million people in 2050. In 2015, the worldwide estimated economic impact of dementia was roughly US$818 billion. While there are multiple pathological brain processes that can culminate in dementia, Alzheimer’s disease (AD) accounts for 60 – 80% of all dementia cases making it imperative to understand the root causes.
In 1901, Alois Alzheimer observed a patient at the Frankfurt Asylum named Mrs. Auguste Deter. The 51-year-old patient had strange behavioral symptoms, including a loss of short-term memory. In April 1906, Mrs. Deter died and Alzheimer had the patient records and the brain sent to Munich. He identified amyloid plaques and neurofibrillary tangles which represented the first time the pathology and clinical symptoms of presenile dementia (later renamed Alzheimer’s Disease; AD) were presented together.
Since that initial discovery, much has been gained with respect to the cellular pathologies indicative of AD. Accumulation of the beta-amyloid protein (β-amyloid, or A-beta [Aβ]) outside neurons results in the formation of amyloid plaques (protein aggregates). Aβ is formed by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase. Cleavage of APP by β-secretase and γ-secretase releases the Aβ fragment and under suitable conditions (high concentration, oxidizing environment), begins to self-aggregate. Aβ aggregates can induce membrane lipid peroxidation (MLP), which impairs the function of membrane ion-motive ATPases (Na+ and Ca2+ pumps) and glucose transporters making neurons and synapses vulnerable to degeneration.
A second pathological feature of AD occurs when tau proteins are hyperphosphorylated causing it to dissociate from microtubules. The hyperphosphorylated tau proteins accumulate in the axoplasm and eventually aggregate into paired helical filaments, coalescing to form tangles. These insoluble aggregates, called neurofibrillary tangles (NFTs), form and destabilize microtubules ultimately disrupting axon function. As the microtubules break down, axonal transport of vesicles and other organelles to the synapse becomes disrupted, leading to impaired synaptic function.
While these pathological features have been intensively explored, there are newly discovered molecular processes at work that culminate in the neurodegenerative and behavioural features indicative of AD. This ebook presents these newly discovered molecular pathways that are at play during the neurodegenerative progression underlying AD. Developing an appropriate animal model of this human disease is of critical importance and the appropriate design of a valid animal model may be key in gaining a more complete understanding of the complexities of the disease process as well as in the development of therapeutics aimed at slowing, daresay, reversing the progression of the degeneration. This ebook also presents an update on the structural changes that are associated with AD pathology including synaptic and neural changes as well as alterations in lipid composition that may be a key mediating factor in the over-all deterioration of the brain. The final four chapters provide an update on how the emergence of plaques and tangles can lead to neurodegeneration with a focus on membrane breakdown, impaired intracellular transport, oxidative stress and calcium dysregulation.
With a clearer picture of the molecular cascades that mediate the pathological processes associated with AD, we will be in a better position to develop more effective therapeutics in staving off this ever-increasing global disease. This will not only have beneficial outcomes at the global level but also at a more personal level enhancing quality of life for the elderly and those that take care of them. This ebook aims to update our understanding of the molecular pathology of AD in hopes of providing the foundation for an effective cure.
List of Contributors
Differences and Implications of Animal Models for the Study of Alzheimerʼs Disease
Marcela Bermudez Echeverry1,2,*,Sonia Guerrero Prieto1,João Carlos dos Santos Silva1,Maria Camila Almeida1,Daniel Carneiro Carrettiero3Abstract
In behavioural neurosciences, animal models are aimed at providing insights into normal and pathological human behaviour and its underlying neuronal processes. Alzheimerʼs disease (AD) is the most common origin of dementia in the elderly. Several factors have been identified, such as the amyloid precursor protein (APP), hyperphosphorylation of tau protein, and the secretase enzymes. Animal models are important for elucidation of mechanistic aspects of AD. Transgenic models recapitulate expression of human β-APP and tau hyperphosphorylation to understand the pathogenesis of AD. In this chapter, some animal models are reviewed and discussed briefly in order to elucidate some criteria that an animal model should fulfil to mimic human neurodegenerative diseases.
INTRODUCTION - ALZHEIMERʼS DISEASE
The chronic or progressive dysfunction of cortical and subcortical function that results in complex cognitive decline that extends beyond normal cognitive decline, is usually described as dementia. These cognitive changes include symptoms affecting memory, impaired judgment or language, thinking, and are commonly accompanied by disturbances in social abilities that may eventually result in impairment of daily functioning, such as becoming lost at a usual path, or paying bills (Mayo Clinic on Alzheimer's Disease). Alzheimer’s disease (AD),
frontotemporal dementia (FTD), dementia with Lewy bodies, are usually referred as primary degenerative dementias, while secondary dementia might be the ones that occur as a consequence of another disease process [1].
Overall dementia incidence increases with age. For instance, 1.5% prevalence in patients over 65 years is described in developed countries, with the ratio doubling every 4 years reaching 30% of prevalence in patients with 80 years [2], being lower in men and in individuals of African or Asian origin [3]. Life expectancy is substantially shortened in dementia patients with 8 years of survival in average from the time of the diagnosis [4], with longer survival for women patients with AD and vascular dementia [5]. It is expected that the number of people living with dementia will double every 20 years, reaching 115.4 million by 2050 [6], being estimated that by 2050, 1 in 85 adults will be diagnosed with AD and a new case of AD is expected to develop every 33 seconds [6].
Current treatments with Acetylcholinesterase inhibitors (AChEIs) and memantine are well established in AD, but the effectiveness of the treatment varies across the population. Nevertheless, current drugs help mask the symptoms of AD, but do not treat the underlying disease or delay its progression, which makes the new developments to treat AD as an important topic. On the other hand, AChEIs use in the daily clinical routine cannot be recommended, with severe side-effects in patients with ʻfrontal lobe dementia’ or FTD [7]. Extensive exploration of possible risk factors could give some clue, but so far no conclusive result has been obtained. Fig. (1) shows main risk factors identified in AD [8].
Fig. (1)) Hypothetical scenarios for the onset of Alzheimerʼs disease (modified from [9]).Risk Factors
• Alcohol Use - There is uncertainty under the effect of alcohol consumption and the incidence of dementia and cognitive decline [10]. Moderate amounts of alcohol has been shown to have a protective effect, however the risk of developing dementia may increase due to alcohol abuse [11].
• Atherosclerosis - It is a fairly common problem associated with aging. The stroke and reduced blood flow as a consequence of accumulation of fats, cholesterol and other substances on artery walls can also cause vascular dementia. Besides, studies have shown that conditions associated to blood vessels (vascular, stroke) may also be associated with AD [12].
• Blood Pressure - Both low (associated with hypometabolism) and high blood pressures have been quoted to serve as factor risk for dementia development [12, 13].
• Depression - Mood alterations may lead to cognitive dysfunctions and specially in men, late-life depression can be a indicative of dementia development [12, 14].
• Diabetes - An increased risk of developing AD and vascular dementia is associated with diabetes [12]. Furthermore, depending on their medial temporal lobe atrophy, long-term users of insulin have been shown to present significantly increased levels of plasma Amyloid β (Aβ) [15].
• High Estrogen Levels - Estrogen-alpha receptor is highly expressed in hippocampus, basal forebrain and cerebral cortex. Several studies have showed mechanisms underlying estrogen neuroprotection in cellular culture, consistent with a lower risk of AD described for women treated with steroid hormone estrogen compared to those who had not [16]. However, AD and depression has been shown to be affected by estrogen levels. Besides, a greater risk of developing dementia has been described for women on estrogen and progesterone replacement treatment for menopause [17]. Contradictory research findings have raised the hypothesis that there is a critical period during the perimenopause or just after menopause, in which hormonal replacement therapy could exert cognitive benefits [16].
• Homocysteine Blood Levels - Homocysteine, a type of aminoacid produced by the body has been described to increase the risk of developing vascular dementia [18].
• Obesity - The risk of developing dementia at older ages seems to be increased in obese and overweighed individuals at middle ages [19].
• Smoking - Both dementia and vascular diseases risk increase due to smoking [12]. A probable nicotine protected effect is with treatment delivered separately from tobacco, and activation of the alpha7 nACh receptors [20]
• Aging - Aging is for sure one of the major risk factor for AD. Aging is generally associated with telomere and HTERT (human telomerase reverse transcriptase). Functionally, the telomere, a sequence of DNA chains protects the end of chromosome from deterioration. Telomere length seems to be a possible cause for AD, where its shortening plays an important role in cognitive impairment, and pathogenesis of AD involved with oxidative stress and inflammation [21]. With advancing age, HTERT methylation frequency is also described to be involved in the AD pathogenesis [22] with decreased gene expression in CA1 hippocampal region [23], a neuronal population vulnerable in AD. Besides, the process of human aging also comprises an array of changes associated to metabolic state and thermal homeostasis, which has also been suggested as factors contributing to development of AD.
• Cerebrovascular Damage - While clinical findings between vascular dementia and AD are distinguishable, AD-associated cognitive decline can emerge after acute or chronic ischemia, hemorrhagic stroke or hypoperfusion episode, resulting in accumulative oxidative stress mediating neuronal and glial insults [24]. Thus, cerebrovascular pathology affecting the CNS could progress to a cognitive decline, taking several years to impact in the performance of cognitive functions, where the impairment of brain blood irrigation can superimposed on the mild cognitive impairment syndrome viewed as prodromal stage of AD.
• Epigenetics - A significant decrease of global DNA and RNA methylation have been described specifically in entorhinal cortex layer II of AD brain samples [25], and a loss of methylation control of two important genes, BACE and presenelin 1, is directly involved in AD [26]. Molecular derangements in methylation stabilizing factors were identified and associated with neurodegeneration, in special PHF1 (paired helical filaments) and PS396 immunoreactivity, both considered markers for neurofibrillary tangle formation [25, 27]. In addition, a disturbance of cell cycle events, with aberrant re-entry of neurons into the cell cycle (e.g. apoptosis) was also observed in AD [25].
• Genetics - The most prevalent form of AD, known as late-onset or sporadic AD, occurs later in life, with no evident inheritance pattern. However, a risk factor gene identified so far for sporadic AD is the apolipoprotein E (apoE), specifically ApoE4 with a ~16% prevalence in AD patients. Familial AD or early-onset AD, which is rarer (less than 1% of the total number cases), usually starts at age 30-60, is an autosomal dominant mutation with three genes identified: APP, presenilin 1 (PSEN1) and presenilin 2 (PSEN2).
AD involves severe neuropathological changes in the hippocampus, followed by the association cortices and subcortical structures, including the amygdala and nucleus basalis of Meynert [20] (Fig. 2).
Fig. (2)) The nucleus basalis of Meynert innervates the entire cerebral mantle with prominent afferents to limbic areas such as the hippocampus, entorhinal cortex and amygdala.Synapse loss and massive neuronal cell death are characteristics of the AD brain, as well as Aβ (also known as Abeta) plaques and neurofibrillary lesions. The neurofibrillary lesions, also described as neurofibrillary tangles (NFTs) are characterized by hyperphosphorylated aggregates of the microtubule-associated protein tau (Fig. 3A) and can be found in cell bodies and apical and distal dendrites, as well as in the abnormal neurites that are associated with some Aβ plaques [20, 28]. Aβ peptides are typically ~ 4kDa β-pleated sheet peptides with different N- and C-terminal endings that are derived from amyloid precursor protein (APP). β-secretase cleaves the APP, via the endosomal-lysosomal pathway to generate the amino terminus of Aβ [29]. γ-secretase further process the peptide at positions 40, 42, and 43 to generate the Aβ peptide [30] (Fig. 3C). Different N-terminally truncated Aβ has been detected in post-mortem AD brain tissue [31].
Neuropathological diagnosis is confirmed by the presence of neurofibrillary tangles and neuropil threads [36]. The propagation of the disease can be classified into six different stages depending on the location of the tangle-bearing neurons and the severity of changes (transentorhinal stages I-II: clinically silent cases; limbic stages III-IV: incipient Alzheimer's disease; neocortical stages V-VI: fully developed Alzheimer's disease) (Fig. 4).
Fig. (3)) Hallmarks of AD and pathological features. (A) Hyperphosphorylated tau dissociates from microtubule (MT)-associated protein tau, causing them to depolymerize, while tau is deposited in aggregates such as neurofibrillary tangles (NFTs). (B) Graphical representation of the distribution of high and low tau levels, associated with NFTs (intra-neuronal), and Aβ. This picture shows the relation between amyloid plaques and Tau-pathology. (C) The major protein component of the plaques is a 40–42 amino acid polypeptide termed Aβ (Aβ40 and Aβ42), that is derived by proteolytic cleavage from the amyloid precursor protein, APP. Β-Secretase activity has been attributed to a single protein, BACE, whereas γ-Secretase activity depends on four molecules, presenilin, nicastrin, anterior pharynx-defective 1 (APH1) and presenilin enhancer 2 (PEN2). γ-Secretase dictates its length, with Aβ40 being the more common and Aβ42 the more fibrillogenic and neurotoxic species (modified from Götz and Ittner, 2008 [32]). On the other hands, cognitive decline in humans is not proportional to Aβ plaque load [33, 34], but does correlate with soluble Aβ species [33, 35]. Fig. (4)) Temporospatial spreading of tau-positive neurofibrillary lesions (tangles) in the process of AD and Amyloid plaques-positive lesions. According to the study [37], stages I–II refers to alterations mainly confined to the upper layers of the transentorhinal cortex (transentorhinal stages). Stages III–IV presents a severe involvement of the transentorhinal and entorhinal regions, with a less severe involvement of the hippocampus and several subcortical nuclei (limbic stages). Stages V–VI presents a massive development of neurofibrillary pathology in neocortical association areas (isocortical stages), and a further increase in pathology in the brain regions affected during stages I–IV. The red areas are proportional to the severity of tau pathology (modified from [37]).ANIMALS MODELS OF AD – DIFFERENCES AND IMPLICATIONS
“An animal model with biological and/or clinical relevance in the behavioral neurosciences is a living organism used to study brain–behavior relations under controlled conditions, with the final goal to gain insight into, and to enable predictions about, these relations in humans and/or a species other than the one studied, or in the same species under conditions different from those under which the study was performed” cited in [38].
The most thoughtful challenges faced by medical research can be at least partially solved with the help of the tools like animal models. Indeed in dementia research, animal models have become crucial tools [28]. The following definitions found on the web emphasize two important attributes of a model: the open-source platform Wikipedia states that an animal model is “a non-human animal that has a disease or injury that is similar to a human condition” [39]. This definition illustrates that models are valuable as they represent a certain stage of disease and because processes that lead to that stage can be monitored longitudinally. In AD research, animal models have been useful in dissecting the pathogenic mechanisms of the pathology, as well as preclinical drug development. Yet, AD includes several aspects that still require a better biochemical and molecular characterization, and therefore it is imperative to develop tools in order to facilitate translational research.
In the web, Research models (http://www.alzforum.org/research-models) contain information about various animal models of Alzheimer’s disease (see http://www.alzforum.org/res/com/tra) with causative genes as well as other proteins involved in the pathogenic process.
Invertebrate Models
Different animal models have been used to study neurodegenerative diseases. The majority genes of AD are evolutionarily conserved in simple organisms such as Drosophila and C. elegans, allowing the manipulation of orthologous genes and pathways in vivo to better understand of pathogenesis of AD and others disorders.
Caenorhabditis Elegans
It is estimate that at least 83% of the nematode C. elegans (Caenorhabditis elegans, Fig. 5) proteome have human orthologous proteins [40], being an important model to study aging, protein aggregation (proteotoxicity) and other issues. For instance, similarity between the nematode and mammalian aging includes muscle atrophy and lipofuscin accumulation [41].
Fig. (5)) Image of C. elegans adult hermaphrodite.Other charactheristics of the nematode that are considered an advantage in biological research includes high fertility, a short life span and low cost maintenance. On the other hand, the worm does not present the complex behavior and cognitive responses that can be evaluated in vertebrates or mammalian models. Nevertheless, the nematode is considered a good model for the study of molecular pathways in neurodegenerative disease.
In C. elegans, the apl-1, is orthologous to the human APP involved in AD. However, it does not produce the Aβ peptides because it lacks the cleavage by β-secretase [42]. On the other hand, in the transgenic model of C. elegans expressing human- Aβ, accumulation of Aβ3-42 peptide [43] and Aβ1-42 [43] lead to progressive paralysis in the worm [44]. In addition, it has been found three presenilin genesin C. elegans: sel-12, hop-1 and spe-4. The sel-12 and hop-1 mutation results in memory deficits and morphological alterations in cholinergic interneurons [45].
Although complex behaviors are not possible to assess in C. elegans, motor behavior can be tested by three simple analyses: 1) “Chemotaxis”, which corresponds to the movement of crawling in the presence of food stimuli. 2) “Trashing” or the swimming that the nematode exhibit in a liquid medium. 3) “Pharyngeal pumping”, which is the muscular contraction as a result of food ingestion [46]. In addition to motor behavior, simple cognitive functions such as memory and learning are easily assessed in the C. elegans model, by looking at the negative olfactory associative conditioning or long-term associative memory [47] (Fig. 6).
Fig. (6)) The enhancement of avoidance behavior after the exposure to odorants in C. elegans (modified from experimental design [48]).Drosophila
Genetic research has been using the fruit fly (Drosophila melanogaster) for more than hundred years. It was the first organism with fully sequenced genome [49]. The Drosophila is able to provide important insights into for experimental studies of multicellular organisms in genetic, anatomic and behavior fields [50]. The anatomical organization of the Drosophila brain (proto, deuto, and tritocerebrum) is homologous with the human brain, which is divided into forebrain, midbrain and hindbrain [51].
The main advantage of Drosophila is the possibility of gene manipulation. Besides, the short lifespan, with average of 120 days depending of stress or others stimulus, also makes this model an interesting one [50] (Fig. 7). The range of possible behaviors possible to study in Drosophila includes olfactory learning, grooming, courtship, aggression, circadian rhythms and locomotor behavior [48].
Fig. (7)) Drosophila melanogaster with brick-red eyes and transverse black rings across the abdomen. Males are slightly smaller than female with darker backs and easily distinguished from females based on a distinct black patch at the abdomen [52].About 70% of human genes responsible for diseases in humans are conserved in the Drosophila, including the orthologue of the human APP protein, the Appl, which is mainly localized in the cortical region of the fly´s brain [53] (Fig. 8). An orthologue of the α-secretase, the kuzbanian gene, the homologous of BACE, the dBACE, and the functional orthologue of TAU, the dtau have also been described [43]. Additionally, the fruit fly also presented the ɣ-secretase complex.
Fig. (8)) Schematic illustration of proteins in human (A) and orthologous in drosophila (B) correlated with AD (modified from [54]).The drosophila APP orthologous, Appl, have similar domains with the APP of vertebrate organisms, by deleting the Appl gene in the drosophila, researchers have found flies viable, fertile, and morphologically normal, yet they exhibit subtle behavioral deficits [54]. Furthermore, the overexpression of the β-secretase protein responsible for the cleavage of Appl, results in Aβ-like fragment aggregates leading to toxicity and neurodegeneration [55].
In a triple transgenic Drosophila model, combining expression of human APP (hAPP), human β-secretase (hBACE) and Drosophila ɣ-secretase presenilin (dPsn) [56, 57], it was observed Aβ40 and Aβ42 aggregation and the formation of plaques. These flies presented age-dependent neurodegenerative processes such as degenerated axons projections and increased in early fatality rate [58]. Similarly, the Aβ Drosophila transgenic, also presented accumulation of Aβ40 and Aβ42 peptides in the fly brain [59]. It has also been reported that the co-expression of presenilin mutants, APP and BACE accelerated the accumulation process of neurotoxic Aβ aggregates in [57].
In addition, studies assessing the mitochondrial loss in the axon resulting from the aberrant phosphorylation of the Tau are also available. Knockdown model has been used to study the Miro and Milton proteins which regulate mitochondrial attachment to microtubules in fruit fly (Fig. 9), showing that mitochondrial loss produced by Milton increases Tau phosphorylation and enhances neurode- generation regulated by Tau [60, 61].
Fig. (9)) Schematic representation of the protein complex that mediates anterograde mitochondrial movement. Miro is anchored to the outer membrane. The association of Milton with the mitochondrion is caused, at least in part, by the interaction of Milton and Miro (modified from [62]).Different behavioral tests can be performed in the Drosophila model. Motor behavior is evaluated using the geotaxis response assay test [57] or climbing assay [63]. These tests allow evaluating the oriented movement toward a gravitational force (Fig. 10A). The principal differences between the two tests are the number of tubes in the apparatus and analyzed results. Another test is the Pavlovian olfactory associative learning [60] (Fig. 10B). The flies are trained with electroshock paired with one odor and after are exposed to a second odor without shock. Subsequently, the learning is measure allowing that flies choose between the two odors. The courtship behavior can also be analyzed [64] by checking the orientation, tapping, wing extension, courtship song, licking and copulation (Fig. 10C).
Fig. (10)) Possible behavioral tests in Drosophila. (A) Oriented movement toward a gravitational force. The flies that express Aβ cannot fly against gravity force. (B) Pavlovian olfactory associative learning. (B1) The flies are exposed to electroshock and odor, and after are tested with other odor without electroshock stimuli. (B2) The drosophila chooses the preferential odor. (C). Courtship behavior is evaluate by looking at features such as orientation, tapping, wing extension, courtship song, licking and attempted copulation (modified from experimental design [55]).Vertebrate Models
Rodents are the dominant model for the study of AD, but non-mammalian organisms also have been used to investigate neurodegenerative diseases. One example is the zebra fish.
Zebrafish
The zebrafish has the genome fully characterized, and is described to share 50- 80% homologous genes with human sequence [43]. The rapid development, large productivity capacity, easy and cheap maintenance are some of the major advantages of this model besides the facility to introduce genetic changes and the easiness to observe the changes. Although this model has lower cognitive behav- ior than rodent model, the zebrafish shows conditioned responses, memory and social behavior. In addition, their transparent embryos enable the manipulation of genes and proteins, allowing observing the embryogenesis and development of central nervous system (CNS) (Fig. 11).
Fig. (11)) Zebrafish, an animal model using in research. Twenty-four hours post fertilization (hpf), CNS of zebrafish embryo was stained with laminin antibody which outlines the neural tube in green and counterstained with propidium iodide to label the nuclei in red. MHBC: midbrain-hindbrain boundary constriction (modified from [65, 66] and https://www.nc3rs.org.uk/news/five-reasons-why-zebrafish-make-excellent-research-models).The Zebrafish orthologous genes to human known involved in the AD are shown in Table 1. The appa and appb have differential expression patterns in the embryonic period with the appb mRNA found in telencephalon, midbrain, hindbrain, spinal cord and dorsal aorta [67]. On the other hand, the appa expression is observed in telencephalon, ventral diencephalon, terminal ganglia, lens, otic vesicles and somites [68].
One experimental approach used to induce the aggregation of Aβ in the Zebrafish is the hindbrain injection of Aβ-42 peptide in embryos. The Aβ injection results in specific cognitive deficits and increased of tau phosphorylation by GSK-3β [68]. However, no neurofibrillary tangles neither apoptosis markers are found, suggesting that this model can be used to study the early stage of AD, as it presents similar molecular markers found in human. Additional, in the literature there are studies showing that application of Aβ in the eyes of the zebrafish, results in blood vessel branching, suggesting that the Aβ have physiological effects in capillary density [69].
Transgenic zebrafish has been used for study the mechanism of the AD and other neurodegenerative diseases. The expression of green fluorescent protein (GFP) from appb promoter was found in the CNS and vascular tissue in zebrafish during development stages and also in the adult [67], suggesting that this model might elucidate the mechanism in APP gene expression in AD. The overexpression of the GPF with the ɣ-glutamylhydrolase (ɣGH) has been used as a tool to study the hypotheses of oxidative stress in the AD. The folate deficit can induce aggregation Aβ and phosphorylated Tau [70], suggesting a pathological mechanism that connect the Aβ and Tau pathways.
The transgenic human protein TAU-P301L in zebrafish neurons provided an important model, as it resulted in a fast neurodegeneration [71]. Moreover, GSK3β inhibitors treatment reduced the hyperphosphorylation of Tau in this transgenic zebrafish, providing an insight on a tool for the pharmacological assessment.
The presenilin 1 and 2 have been studied in zebrafish model by inhibiting translation of psen1 and psen2 mRNA through injection of antisense oligonucleotides. The inhibition of psen1 resulted in somitogenesis defect [72]; while inhibition of psen2 revealed an important role of this gene in the signaling and embryo development [73], and programmed death cell.
Knockdown is also possible in Zebrafish. In the literature, this technique allowed to demonstrate that appb is necessary for the axonal growth in motor neurons and cytoskeletal of embryos [74].
Regarding behavioral tests possible with the Zebrafish model, the object recognition memory is widely used [75]. To evaluate object preference, the zebrafish is acclimated to the environment and exposed to two identical objects at first and then to a different object, and, finally is exposed to a familiar and a novel object (Fig. 12).
Fig. (12)) Novel object preference test in Zebrafish. Test Object recognition memory. The first step is the acclimatization (5 min) in an identical experimental environmental. After, the fish is exposing to two identical objects for 10 min. During the inter-trial, the zebrafish is put back to the initial tank, and finally, put in the experimental tank for 10 min while exposed to a familiar and a novel object (modified from experimental design [75]Another cognitive function that can be evaluated in zebrafish model is avoidance response [68]. This test consists of a visual stimulus (for example a ball) travelling in the half of tank (stimulus area) (Fig. 13). The number of fishes that are found in the non-stimulus area is considered that have cognitive avoidance ability. The locomotor activity can be evaluated in the zebra fish model through the exploration of new an environment (Fig. 13). The total distance travel, speed, time of mobile and absolute turn angle are possible parameters to be analyzed in this test [68, 76].
Fig. (13)) Escape and avoidance behavior in zebrafish. Avoidance: a potential threat is anticipated by zebrafish that swim away to safety, by swimming to a protected area, diving to the bottom of the tank or joining a shoal of fish. Once the fish relax, they start to explore their surroundings. This way, quantitative data can be obtained for escape, avoidance, and exploratory behaviors (modified from experimental design [68])Rodent Models
The whole aspects of the disease spectrum cannot be recapitulated by a single model. Nevertheless, each model are suitable for in-depth analysis of one or two components of the disease, which is not readily possible or ethical with human patients or samples [34].
Not only the cholinergic, beta-amyloid, and tau theories but also a genetic basis accounts for the multifactorial and complex molecular mechanisms of AD as well as the different symptomatology processes activation among human population which will progress into divergent neuropathological characteristics underlying behavior and cognitive changes [77]. Genetic models have still been invaluable in determining the molecular mechanisms of familial AD (fAD) and molecular events similar with idiopathic or sporadic form of AD.
Study of AD Risk Factors in Rodents
The autosomal-dominant early-onset familial AD are a consequence of mutations that occur in the presenilin 1 (14q24.3), presenilin 2 (1q31-q42), and Aβ A4 precursor protein (APP; 21q21.3) [78], which results in premature onset for the production of soluble 40 or 42 amino acid Aβ peptide. Besides, inheritance of apolipoprotein E epsilon 4 (ApoE4; 19q13.2) allele constitutes a major genetic risk factor (Fig. 14) for developing Early Onset AD as well as late-onset AD predisposition and hypercholesterolaemia. Even though this feature is not sufficient to cause the disease by itself [77], together with other factors such as by acting in synergy with other susceptible genes, for example, programmed cell death protein 4 (PDCD4) and in a complex interaction with environmental factors, could lead to disease development [79].
Fig. (14)) Apolipoprotein E4 (ApoE4) and interaction with Aβ peptide to cause cognitive decline and neurodegeneration, as well converge on the amyloid. Also, ApoE4 can show a complex interaction with environmental factors (modified from [80]).In transgenic mice and in vitro, the ApoE4 could be involved in cholesterol transport hindrance (Fig. 15), diminished neuronal repair, Aβ deposition, fibrillisation, and plaque formation by acting as an Aβ interacting pathological chaperone [81, 82]. ApoE mRNA is found in cortical and hippocampal neurons in humans [83] and in transgenic mice expressing human ApoE under the control of the human ApoE promoter [84].
Fig. (15)) ApoE isoforms in the CNS. ApoE is synthesized by astrocytes, activated microglia, and neurons express apoE under physiological and pathological conditions. Roles for apoE4: (1) enhanced Aβ production, (2) potentiation of Aβ1-42-induced lysosomal leakage and apoptosis, and (3) enhanced neuron-specific proteolysis resulting in translocation of neurotoxic apoE4 fragments in the cytosol, where they are associated with cytoskeletal disruption and mitochondrial dysfunction. Once produced APOE by astrocytes, or activated microglia or under pathological insults, neurotoxic effects are thought to be downstream of APOE 3 – APOE4-mediated toxicity (modified from [80]).In the human and animal brain, staining using Thioflavin S but especially Congo red is the gold standard for diagnosing amyloid plaques because it only binds aggregated β- Sheets [34].
A simultaneous occurrence of hypocholinergic tone and Aβ accumulation in which Aβ and apolipoprotein E epsilon 4 (ApoE4) could interact with alpha7 nicotinic receptors leading to the repression of glycogen synthase 3β and downstream effects towards tau protein hyperphosphorylation has been hypothesized [76, 85]. Indeed, the first theory proposed to explain the etiology of AD was the cholinergic depletion hypothesis. This theory was founded on the studies showing that memory impairment, due to loss in cholinergic transmission, could be reversed following treatment of mild-to-moderate patients with cholinergic receptor agonist (e.g., nicotinic acetylcholine receptors and muscarinic acetylcholine receptors), acetylcholinesterase inhibitors (e.g., galantamine, donepezil, and rivastigmine), acetylcholine precursors (e.g., L-alpha glyceryl- phosphorylcholine), and cholinergic enzymes (e.g., choline acetyltransferase) [76, 86, 87].
Enzymes such as phospholipase A2, involved in lipid membrane metabolism connecting the cholinergic and glutamatergic systems could play a role in cognitive decline and neurodegenerative process in AD. Indeed, inhibition of phospholipase A2 both Ca+2 dependent but also independent can lead to formation of Aβ through downregulation of cholinergic and glutamate receptors. Nonetheless, if Aβ is already elevated during the AD, it could favor upregulation of Ca2+-dependent phospholipase A2 and secretory phospholipase A2 implicated in inflammatory cytokines and oxidative stress [76, 88].
A “partial” model that provides relevant insights regarding the pathological related events listed above is achieved through amyloid precursor protein (APP) mutation transgenic models. Those animals usually show behavioral impairment before deposition of amyloid, and even once an Aβ has developed, neuronal loss tends to be minor [88, 89]. It is thus difficult to analyze how much the cognitive deficits seen in these mice are equivalent to the ones seen in humans. Thus, from a clinical perspective, findings in the animal models may be of interest in terms of understanding brain function but are of dubious significance in terms of understanding a specific human disease. Nonetheless, the partial nature of the model may also be a tool for furthering understanding of the elements important in disease.
In particular, in rodent models, APP, tau hyperphosphorylation, and the secretase enzymes, have become the pivotal point of current investigation. Several transgenic and non-transgenic animal models have been developed to clarify the mechanistic aspects of AD and to validate potential therapeutic targets.
The Rat Model
The rat has also been used as a model for AD pathogenesis. In particular, the effects of cortical Aβ accumulation on the cholinergic, noradrenergic and serotoninergic systems were extensively studied using rats [90, 91]. The AD-like brain insulin signaling impairment in rats are studied using the intracerebral injection of streptozotocin- icv (STZ) [90, 92]. These animals display reduced choline acetyltransferase activity [93], with high levels of oxidative stress and nitrative stress [94], astrogliosis, several inflammatory processes, and axonal neurotoxicity [95], changes in brain insulin signaling and alterations in Aβ homeostasis and higher levels of hyperphosphorylated Tau [96], as well as cognitive impairments as featured by deficits in learning, memory and cognitive behavior [96]. This model can represent sporadic AD.
Cerebral microinjection of a vehicle containing oligomers and Aβ monomers but not amyloid fibrils are commonly also used as a rat model for AD studies, as those injections have been shown to inhibited hippocampal long-term potentiation (LTP) in rats [97].
The Rabbit Model
Rabbits have become useful for modelling AD when validating the neuroprotective effects of metal chelators [98]. Adding copper to a group of cholesterol-fed rabbits can induce amyloid deposition and cognitive impairment, with Aβ aggregation related to redox activity of metals such as copper [99].
The role of Aluminum (Al) linked to AD remains controversial and the hypothesis has been abandoned by researchers. Previously, in 1965, Wisniewski, Terry, and Klatzo demonstrated the formation of NFTs after brain injection of Al into rabbits [100]. Al3+ administration also has been described to influence oligomerization and promotes conformational changes in Aβ, suggesting an effect on amyloid cascade [101]. Nevertheless, it was verified that excessive oral intake of Al does not accelerate AD pathophysiology in transgenic mouse for Aβ and tau phosphorylation accumulation (AβPP and AβPP/tau transgenic mice) [102], as such, in other AβPP mice model, Tg2576 mice, the start of plaques deposited at 9 months of age was also not modified after administration Al via diet from 6 to 9 months of age [103]. Thus, Al is associated with dialysis encephalopathy that differs from AD in presentation, clinical progression, and time course [104]. Yet, despite this clinical picture difference, Al is considered neurotoxic compound and it can induce decreased expression of neurofilament [105], neprilysin [106] and altered expression of β-APP secretase (BACE1 and BACE2) [107].
Transgenic AD models have been generated to investigate FAD mutation, thus the phenotype of transgenic strain, promoter used and the background mouse strain are very important points to consider when choosing a transgenic models to study AD clinical symptoms [33]. In Table 2 and Fig. 16, a summary with the most used transgenic mouse models.
Transgenic Mice
The discovery that tau dysfunction and Aβ accumulation is the essential events leading to neurodegeneration in AD have led to the development of transgenic animal models. Most transgenic rodent models are based on the over-expression of three mutated genes: (i) amyloid precursor protein (APP) and/or (ii) Presenilins (PS1 and 2) and/or (iii) tau hyperphosphorylation and formation of NFTs (MAPT – microtubule-associated protein tau).
Götz & Ittner [32] had shown a schematic representation to study AD and FTD (Fig. 16). FTD appear between the ages of 40 and 65, usually does not include formation of amyloid plaques, exhibit impolite and socially inappropriate behavior and is linked to a mutation in the tau gene FTDP-17, N279K, ΔK280, P301L, P301S, V337 and R406W [32]. Consistent with patients expressing early onset of FTD, P301S, where mouse prion protein (PrP) was used, it shows higher neuronal loss than in P301L tau transgenic mice, with changes prominent in brain areas and ventricular enlargement, as observed in patients with FTD [108].
Fig. (16)) Expression of plaques and NFTs in transgenic mice. Mutations are listed (grey boxes) together with their strain names and the promoters (in parenthesis) that were used for expression. Here, progression of the pathology in pR5 mice showed NFT formation in the amygdala and eventually found in the hippocampus, but the cortex is virtually spared. In APP23 mice, plaque formation is protuberant in the cortex and in the hippocampus. FTD, usually does not include formation of amyloid plaques and tau transgenic mice are used. This reflects, to some extent, the situation in the brain of patients with AD, in which plaques and NFTs are anatomically separated (modified from [32]).The most commonly used transgenic mice in AD to study different neuro- pathological changes are challenged to behavioral test to assess hippocampus-dependent memory functions. Since AD is a neuropathology accompanied by cognitive failure, behavioral tests are critical requirements with well-established hippocampal-dependent learning and memory assessment correlates with biological mechanisms like resources for drugs discovery.
Overexpression of the Amyloid Precursor Protein (APP)
PDAPP mouse is generated from platelet derived growth factor-β (PDGF) promoter plus APP, which results in an 18-fold elevation of APP RNA and age-dependent amyloid deposition, with dystrophic neurites and reactive astrocytes. Aβ42 immunotherapy studies in young PDAPP mouse, resulted in amyloid pathology reduction in older mice, with ameliorated memory deficits [109]. However clinical trials of the same immunotherapy resulted in meningoencephalitis development in some individuals, even though a reduction in Aβ plaques and astrogliosis was observed in some areas of cortex [110].
Like PDAPP mice, the Tg2576 mice, the most widely transgenic model used, express a 5-fold increase in human APP levels in cortical and limbic structures, with spatial memory impaired in 9- to 10- month-old mice [111]. This transgenic express increased Aβ40 and Aβ42 more or less proportionately.
After PDAPP and Tg2576 mice many other transgenic lines were developed with difference in region and temporal profile of plaque deposition. TgCRND8 mice expressing multiple APP mutations, exhibits amyloid deposition at 3 month of age. APP23 mice is used because the Thy-1 promoter results in prominent vascular amyloid deposition. Similarly, Tg2576 mice also show huge vascular amyloid plaques, whereas this feature is absent in PDAPP mice [112].
Other difference among APP transgenic is the genetic background. PDAPP mice have been maintained on a mixed C57BL6/DBA/Swiss-Webster background, whereas, Tg2576 mice are studied on a hybrid C57BL6/SJL background [113].
Presenilin Mutations Transgenic
Mutations in PS1 is the most recognized cause of early-onset FAD, with ~160 mutations already described. PS1 has a locus on chromosome 14, and PS2 on chromosome 1. There is more PS1FAD than PS2 FAD mutant transgenic lines. PS1 FAD has been generated with the same promoters used for PDGF and PrP (APP transgenic mice). Although PS FAD mutant shows increase in Aβ42, single transgenic PS1 or PS2 mice do not develop plaques. However, PS when crossed with APP lines, mutations cause earlier and widespread plaque formation. The PS1/APP mice are frequently used. Besides, PS1 or PS2 FAD mutant lines shown excessive neuronal loss in the entorhinal cortex, with impaired hippocampal neurogenesis, age-related neurodegenerative changes [114, 115]. Combined mutations of 3 APP and PS1 FAD created a 5X FAD mutant mouse with neuronal loss sufficient to cause AD similar to humans [116].
Tau Models and Aβ–tau Association
As aforementioned NFTs containing hyperphosphorylated forms of tau can be studied with thioflavin-S and Congo red histological dyes, and they are recognized by specific antibodies. Although, phosphorylated tau accumulates within dystrophic neurites in PDAPP mice, these alterations cannot be recognized by histological dyes as in humans [117]. Then, the first crossed transgenic line known as JPNL3 to develop NFT-like lesions, expresses the P301L mutation (transgenic line that reproduce aggregation and NFT-formation in mice [118, 119], associated with FTDP-17, and Tg2576 mice. A triple transgenic model, 3xTg-AD, was generated (2 transgenes containing APP-Swedish and P301L FTDP-17 mutations), where mice had increased Aβ40 and Aβ42 levels, accumulated intraneuronal Aβ, amyloid plaques and NFT-like lesions [120]. In addition, in 3xTg-AD mice, amyloid plaques preceded tau pathology evident until about 1 year of age, with altered synaptic dysfunction and deficits in spatial memory [120
