Mutagenesis Advance Access originally published online on December 8, 2006
Mutagenesis 2007 22(1):15-33; doi:10.1093/mutage/gel055
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A review of genome mutation and Alzheimer's disease
1 CSIRO Health Sciences and Nutrition, PO Box 10041 Adelaide BC, Adelaide, South Australia 5000, Australia 2 Discipline of Physiology, School of Molecular and Biomedical Sciences The University of Adelaide, Adelaide, South Australia 5000, Australia
Alzheimer's disease (AD) is a complex progressive neurodegenerative disorder of the brain and is the commonest form of dementia. The prevalence of this disease is predicted to increase 3-fold over the next 30 years and to date no reliable and conclusive diagnostic test exists that will identify individuals presymptomatically of susceptibility risk. This review examines the molecular, genetic, dietary and environmental evidence underlying the known pathology of AD and proposes a biologically plausible chromosome instability model to explain some of the features of the disease. Genome damage biomarkers such as aneuploidy of chromosome 17 and 21, oxidative damage to DNA and telomere shortening together with abnormal expression of APP, ß amyloid and tau proteins are discussed in terms of their potential value as risk biomarkers. These biomarkers could then be used in diagnosis and the evaluation of potentially effective preventative measures.
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Introduction |
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Alois Alzheimer (Figure 1) was born in Marktbriet, Germany on June 14, 1864. He studied medicine at the Universities of Berlin, Tübingen, and Würzberg where he completed his doctoral thesis under the supervision of Albert Kölliker on ceruminal glands in 1887. From 1888 to 1903 Alzheimer worked as a medical resident and then later as a senior physician at the municipal mental asylum in Frankfurt. It was here that he forged his friendship with Franz Nissl, who developed histopathological stains that allowed the histology of nervous tissue from various neurodegenerative disorders to be studied.
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On November 25, 1901 a patient called Auguste D was admitted to Frankfurt hospital where she was seen and treated by Alzheimer. She exhibited various behavioural and psychiatric symptoms including paranoia, delusions, hallucinations and impaired memory (1
). After having suffered 5 years of illness she died in 1906. Her clinical notes and brain were forwarded onto Alzheimer in Munich, where over the next few months he examined Auguste's brain in great detail. At the 37th Conference of German psychiatrists meeting in Tübingen on November 4, 1906, Alzheimer reported for the first time the histopathological changes that he had witnessed in Auguste's brain. In his journal he wrote "in the centre of an almost normal cell there stands out one or several fibres due to their characteristic thickness and peculiar impregnability. Numerous small miliary foci are found in the superior layers. They are determined by the storage of a peculiar substance in the cerebral cortex. All in all we have to face a peculiar disease process" (2). The impregnable fibres so described by Alzheimer were the neurofibrillary tangles, whereas the miliary foci were to be later referred to as the amyloid based neuritic plaques. Both these structures initially described by Alzheimer are now recognized as the characteristic hallmarks of a disease that now bears his name. In 1910 Emil Kraepelin published the 8th edition of his book The Handbook of Psychiatry where he describes a particularly serious form of senile dementia with early age of onset as Alzheimer's disease.
Having worked with Kraepelin in Munich from 1903 to 1912, Alzheimer was appointed to the position of professor of Psychiatry in Breslau, Poland. However with the arrival of the First World War conditions became increasingly more difficult. He found himself under increasing stress until finally his health started to fail. Alois Alzheimer died in a uraemic coma as a result of rheumatic endocarditis on December 19, 1915 at the age of 51. Alzheimer's many years of dedicated research provided the foundation for today's extensive research programmes, into trying to understand a disease that is predicted to make a huge social and financial impact on the 21st Century. Alzheimer's disease (AD) has been classified as a progressive degenerative disorder of the brain and is the most common form of dementia, with between 50 and 70% of all clinically presented cases being histopathologically confirmed as AD at post mortem (3). Worldwide a new case of dementia is diagnosed every 7 s. The global incidence of dementia is estimated to be 24.3 million, with 
4.6 million new cases being diagnosed annually (4,5). Currently between 165 000 and 180 000 Australians suffer from this disease, with an annual cost in 2004 to the Australian government of $3.6 billion dollars in lost productivity and medical care (6,7). These numbers are set to increase 3-fold over the next 30 years as a greater proportion of an already ageing population reaches retirement age. Advancing age is the major contributing factor for increased risk of developing Alzheimer's. After the age of 65 a doubling of risk occurs every 5 years affecting 30% of individuals aged 
80 years (3,8,9) It is estimated that by 2025 at least 34 million people worldwide will suffer from AD (10).
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Clinical diagnosis |
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At present, based upon criteria of cognitive impairment and behavioural changes patients can be clinically diagnosed with an accuracy between 60 and 70% of having AD (11
). The most commonly used criteria are those outlined by the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and-related Disorders Association (NINCDS–AD & DA), published in 1984 (12). According to this document, criteria for probable diagnosis for Alzheimer's includes dementia as determined by the mini mental state examination (MMSE) (13), which allows a brief quantitative measure of cognition status to be determined. It can be used as a measure of cognitive decline, to document cognitive changes with the passage of time and in relation to treatment, and as an effective tool in screening for elements of cognitive impairment. Dementia is diagnosed when two of the following parameters are impaired: amnesia (progressive worsening of memory), aphasia (impairment of speech), apraxia (the inability to perform motor tasks) and agnosia (the inability to identify and recognise individuals or objects, despite having knowledge of the characteristics of those individuals and objects). Individual assessment includes various psychiatric and behavioural changes such as depression, misidentifications, delusions and hallucinations that invariably lead to an individual's inability to perform everyday tasks, resulting in some form of full-time care being required (8).
The age of onset for AD is usually after the age of 65 years but can be earlier if influenced by genetic mutations in familial Alzheimer genes. Other forms of systemic disorders, which could also account for the progressive decline in both memory and cognition function invariably need to be ruled out. Laboratory testing can aid in the identification of conditions that need to be diagnostically eliminated in order to achieve a more accurate diagnosis when a dementia profile is being evaluated. A complete blood cell count and urinalysis profile is necessary to exclude signs of anaemia and infection; metabolic disorders can be evaluated through analysis of serum metabolites (such as glucose, calcium, urea and creatinine), as well as, by performing various liver function tests to eliminate other certain metabolic disorders such as Wilson's syndrome or Laennec's cirrhosis. Additionally, neurosyphilis as well as micronutrient deficiencies in folate and B12 need to be excluded as they can simulate symptoms of dementia, thereby, making an accurate diagnosis more difficult to achieve (14).
Until recently there was no diagnostic test available that could reliably and conclusively identify those individuals for increased presymptomatic risk of AD. Consequently it was impossible to develop and implement effective preventative measures to curb the progress of the disease. However recently a simple non-invasive skin test that measures dilation of blood vessels has been developed, that reflects a decline in function in specific blood vessel cell types affected by the disease. It is claimed that different dementias can be differentiated and that detection can be made up to two years before conventional clinical diagnosis (15,16). A further technique has been developed that involves the use of an intravenous amyloidophilic compound labelled with 19F. This compound when administered to amyloid precursor protein (APP) transgenic mice, specifically labelled amyloid plaques that could be visualized in living subjects by magnetic resonance imaging (MRI). This technique would allow the identification of the disease at an earlier stage in presymptomatic individuals and to determine disease progression in response to various preventative measures and selected treatments (17).
Although the pathogenesis of the disease is still not completely understood, it has been shown that at post mortem there are two histopathological changes that occur within the brain. These changes involve the abnormal clustering of proteins, which characterize individuals with AD. This abnormal clustering occurs in two forms: (i) those that occur within the neurons, i.e. the neurofibrillary tangles and (ii) those that cluster extracellularly outside of the neuronal body, i.e. the amyloid based neuritic plaques (Figure 2). Early changes occur within the entorhinal cortex projecting into the hippocampus, which leads to disruption of learning and short-term memory processes. Further protein deposits have been found within the temporal, frontal and inferior parietal lobes mapping the spread of the disease throughout the brain. Later developments in the pathology of AD include neuronal cell death resulting in loss of tissue leading to overall shrinkage of brain size (10).
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Tau and neurofibrillary tangles |
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Neurofibrillary tangles are composed of the microtubule-associated protein tau, the gene of which is located at chromosome 17q21.1 (18
). The TAU gene comprises of 15 exons, with 11 of the exons coding for all of the major tau isoforms. Splicing of exons 2, 3 and 10 results in the six different forms of the tau protein. These forms differ from each other by the presence or absence of two N-terminal exons and a single C-terminal exon, which results in variation of their amino acid length, which ranges from 352 to 441 amino acids (19).
Tau proteins are important as they associate with tubulin in the formation of microtubules. Microtubules impart shape and structure to cells and generate cellular transport networks that allow movement of micronutrients, neurotransmitters and organelles that are essential for normal cellular function (20). The tau protein of neurofibrillary tangles consists of paired helical threads that are hyperphosphorylated. This hyperphosphorylation leads to a dissociation between tau and tubulin resulting in a breakdown in the brain transport network eventually leading to resistance to protein degradation (20), loss of biological activity and cell death (18,20).
The level of tau phosphorylation is probably determined by the regulation of protein kinases that modify tau through phosphorylation, and phosphatases that dephosphorylate the modified tau. Tau kinases such as GSK3, cdk5 and p38 are known as proline kinases and regulate proline modified serine and threonine motifs. Both Cyclic-AMP and Ca2+/cadmodulin dependent kinases together with protein kinase C are known as non-proline kinases that modify non-proline serine and threonine motifs. Sequences involved in binding tau to tubulin are coded for within the C-terminal exon and are positioned adjacent to the sequences for the tubulin binding repeats (21) (Figure 3).
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Tau phosphorylation is linked to microtubule stabilization by regulating the binding of tau to the microtubules. In AD under conditions following phosphorylation, decreased binding occurs leading to a breakdown in microtubule stability resulting in tau aggregation within the neurons (22
). Hyperphosphorylation may also lead to microtubule spindle defects, resulting in aneuploidy for a number of chromosomes including chromosome 17 that may lead to abnormal expression of Alzheimer's-related genes such as TAU.
In the neurodegenerative disorder frontotemporal dementia, the density of the neurofibrillary tangles is directly correlated with the severity of exhibited dementia (10,23). This disorder is the result of point mutations within exon 10 of the TAU gene such as Leu266Val and Glu342Val. The disorder is not associated with any amyloid peptide formation or plaque deposition (24,25). This is important as it suggests that dementia can arise directly from abnormal processing and accumulation of tau (26,27) that arises independently of any influence of abnormal amyloid metabolism.
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ß amyloid and neuritic plaques |
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The amyloid based neuritic plaque occurs extracellularly to the neuron body having originated within the neuron and been secreted as a soluble peptide. It is thought to be the first histopathological change to occur in AD (10
). The plaque consists primarily of the 42 amino acid ß amyloid peptide (Aß42) originating from the abnormal processing of APP, the gene of which is located on chromosome 21q21. APP is a cell-surface protein of 695–770 amino acids long, and plays a functional role in neurite outgrowth, cell adhesion, synaptic functions and the induction of apoptosis (28).
The protein runs through the membrane leaving a short intracellular terminal and a longer terminal extracellularly (Figure 4) .The ß amyloid peptide is normally snipped out of the APP protein that is adjacent to the cell membrane. APP proteolysis is mediated by a series of secretase enzymes 
, ß and 
(29,30). Under normal conditions, a harmless P3 fragment is formed by cleavage by and secretases resulting in a 40 amino acid ß peptide (28,31).
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The
secretase is thought to be made up of metalloproteases of the tumour necrosis factor -converting enzyme and a disintegrin matrix (32). ß Sectretase has been identified as the enzyme BACE (ß site APP cleaving enzyme) the gene of which is located on chromosome 11. The secretase is a complex multi protein comprising of four components, presenilin, nicastrin, Aph-1 and Pen2 all of which are required for effective proteolytic activity (33).
In the Alzheimer's brain, aberrant proteolysis occurs involving ß secretase which produces a series of products, that after having been cleaved by gamma secretase, gives rise to the Aß42 (29). Aberrant proteolysis results in an abnormal accumulation of soluble Aß42, which aggregates into oligomers. When fibrillized and subjected to an astrocyte induced inflammatory response it matures into an insoluble neuritic plaque measuring between 10 and 120 µm. (28,31,34).
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Intraneuronal Aß42 is generated initially within the endoplasmic reticulum, golgi body and endosomes of the neuron where it accumulates, eventually transferring to the extracellular space of the neuron as a soluble peptide (35
–38). It has also been shown to accumulate in those vulnerable areas of the brain that are sensitive to Alzheimer's-related pathological changes such as the perikaryon of the pyramidal neurons of the hippocampus and entorhinal cortex (39) (Figure 6). Intraneuronal Aß42 could be considered to be an early pathological biomarker of the disease and has been shown to be a contributory factor to neuronal dysfunction.
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Billings et al. (40
) have shown in a transgenic mouse model that animals manifested symptoms of cognitive impairment that correlated with intraneuronal Aß42 accumulation within the hippocampus and amygdala. The mice were free from both tangle and plaque pathology suggesting these structures contribute to further cognitive decline during the later stages of the disease. It may also be that they are incidental endpoint structures that are not directly responsible for the causative factors leading to the pathology of the disease. By clearing the intraneuronal Aß42 using immunotherapy, the observed cognitive deficits were attenuated when memory was assessed after measuring tasks that involve the hippocampus. This implies that the intraneuronal Aß42 has a role in the initiation of early stage cognitive impairment and neuronal dysfunction in AD (40).
It has been shown that application of Aß42 to cultured neurons is neurotoxic and can directly initiate apoptosis (41,42). The presence of ß amyloid elevates apoptotic vulnerability when cells are under conditions of increased oxidative stress, which occurs naturally in the ageing brain. ß amyloid induced oxidative stress results in the generation of reactive oxygen species that can damage various cellular components such as cell membrane proteins, mitochondrial DNA, lipids and cytoplasmic proteins. In the brains of Alzheimer's patients these components have been shown to exhibit elevated levels of oxidative damage (43,44). ß amyloid causes neurons to undergo apoptosis by sensitizing their membranes following lipid peroxidation. The normal ATPases associated with the membrane are impaired leading to membrane depolarization, and a disruption in glucose and glutamate transportation and ATP energy depletion. ß amyloid also modifies calcium flow across the membrane (45). Calcium is involved in mechanisms associated with memory and learning, as well as, neuronal survival. The ß amyloid peptide disrupts calcium regulatory pumps within the membrane and elevates calcium influx through voltage dependent channels, possibly as a result of induced oxidative stress leading in turn to neuronal cell death (46).
Further signs of apoptosis in Alzheimer's brains include elevated DNA fragmentation and ß amyloid induced caspase activation. Caspases are a family of cysteine proteases and are the principal effectors of apoptosis. In Alzheimer's brains caspase 3 has been shown to use APP as a substrate, producing a potent apoptotic promoting peptide called C31 (47). Other caspases such as caspases 8, 9 and 12 have been shown to contribute to neurodegeneration (48,49). Nakagara et al. (49) has shown that caspase 12 located in the endoplasmic reticulum (which regulates cellular responses to stresses such as high calcium and free radicals) induces apoptosis as a result of ß amyloid toxicity. It has been suggested that caspase activation occurs initially at the nerve synapses (50), leading to an overall loss of cerebral nerve terminals and interneuronal communication within the neuronal network. This gradual degeneration of terminals may correlate with the individual's level of cognitive impairment. As nerve cells slowly undergo further terminal loss with the passage of time, the dysfunctional neurons eventually die in those areas of the brain that are initially susceptible to ß amyloid toxicity.
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Amyloid cascade hypothesis: plaques or tangles? |
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One of the topics that has generated a great deal of interest within Alzheimer's research is the question of which of the histopathological landmarks, the neurofibrillary tangle or the neuritic plaque, appears first? Does one influence the development of the other or do they in fact develop independently via two separate pathways? The amyloid cascade theory was proposed to explain the aetiology and progression of the disease (51
–53) and indeed still forms the backbone of explanation for Alzheimer's development. However in recent years it has been found that certain observations cannot be explained by the hypothesis in its current form (54,55). The cascade theory currently states that elevated deposits of the Aß42 occur as a result of missense mutations or a failure of clearance mechanisms that invariably lead to the production of neuritic plaques. These plaques precede intracellular accumulation of tau and eventually result in neuronal cell death (52,53). The hypothesis also suggests that the plaques produce hyperphosphorylated tangles through the abnormal regulation of kinases and phosphatases as the disease progresses (56).
Various lines of evidence have come to light in support of the hypothesis. First, mutations both within the APP and presenilin genes (genes implicated in early-onset AD) result in elevated production and accumulation of the Aß42 (51,57,58). The Aß42 is more sensitive to fibrillization and therefore also more sensitive to neuritic plaque formation (51). Over-expression of the APP gene leads to elevated ß amyloid production resulting in the development of amyloid plaques. Down's syndrome patients possess three copies of chromosome 21, which codes for the APP gene and are therefore susceptible to excessive expression of APP and possibly generation of an excess of Aß42. An identical brain histopathology to Alzheimer's patients is apparent in Down's syndrome patients between the ages of 30 and 40 years (59–61) with large numbers of tangles appearing between the third and fifth decades of life. Second, mutations within exon 10 of the TAU gene give rise to frontotemporal dementia characterized by cognitive impairment but occurring in the absence of plaque formation (10,24,25). This implies that severe tau production and accumulation is sufficient to produce symptoms of dementia independently of amyloid plaques and has little influence on the initiation of amyloid plaque formation. Thus Alzheimer's tangles would appear to be produced after changes in the amyloid production rather than before (51). Transgenic mouse models have been useful in yielding etiological clues to both the progression and underlying mechanisms associated with disease. JNPL3 transgenic mice expressing altered tau when crossed with Tg2576 transgenic mice expressing altered APP showed no change in the number, age of development and morphology of the neuritic plaques produced. However an increase in tau positive tangles was more substantial within the limbic system indicating a role for APP or Aß42 in the formation of neurofibrillary tangles (62). This suggests that changes in the processing of the APP gene occur prior to tau alteration. Additional experiments involving crossing APP transgenic mice with apolipoprotein allele 4 (APOE4) deficient mice resulted in offspring with a decrease in the deposition of ß amyloid, indicating an interaction with the APOE4 gene and amyloid processing (63). Figure 5 outlines a modified version of the amyloid cascade hypothesis proposed by Hardy (52) and Hardy and Selkoe (56).
Although no other model trying to explain the natural history of AD has been put forward, the model in its current form has come under a certain amount of criticism. It has not been able to account for a number of observations that are important in the pathology of AD. One of the main criticisms is that there is only a weak correlation between plaque density and the degree of severity of dementia (64). Additionally, transgenic mice over-expressing the APP gene have been shown to exhibit little or no neurodegeneration (65). Further a small number of cases have been reported involving exon 9 mutations within the Presenilin 1 (PSEN1) gene involving Alzheimer's patients with spastic parapesis (66,67). This condition is unusual in that few amyloid plaques appear within the brains of affected individuals. Further evidence has indicated that the neurofibrillary tangles appear at least a decade prior to the formation of neuritic plaques (68,69). Various studies have shown that tangles were present in areas of the hippocampus and entorhinal cortex in all non-demented patients over 60 years of age, whereas cases that were up to 90 years old were found without plaques (70,71). In their monumental study investigating 2661 cases Braak and Braak (69) found neurofibrillary tangles were evident in the entorhinal cortex in up to 98% of brains examined, whereas only 70% had any evidence of neuritic plaque deposition (69). Further analysis showed that the initial phase of tangle development preceded plaque deposition by up to two decades (72).
The problem is therefore how to explain the substantial evidence supporting the amyloid peptide as a causative agent of Alzheimer's on the one hand, with the equally strong evidence implying that tangle formation precedes plaque deposition and therefore does not fit into the current cascade hypothesis model. It has been proposed that neurofibrillary tangles are an independent feature accumulating slowly with age within the medial temporal lobes. However under the influence of altered amyloid metabolism, which leads to plaque formation during the initial stages of Alzheimer's, there is an acceleration of tangle formation that spreads further to include the neocortex (70). It appears that tangles form normally with age independently of plaque formation. The density of tangles increases with age in an exponential fashion and without any amyloid influence appears to remain confined to the medial lobe (73). As tangle physiology involves synaptic and neuronal loss there would be a stronger correlation between tangle density and degree of severity of dementia. This would be facilitated as soon as tangle acceleration occurs as a result of plaque influence (70). It would appear that since its initial conception the cascade hypothesis has been supported through a whole body of work that has emerged from laboratories from around the world. Alternative models have not been proposed to explain deficiencies within the cascade hypothesis with the same degree of experimental support that is available for the current model. With the passage of time and with the emergence of new experimental data, the current hypothesis will be either reshaped to explain the pathogenesis of the disease or be replaced with an alternative model, which will have to explain all facets of aetiology and progression of the disease.
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Genetics of AD |
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Inheritance of known genes that predispose to AD accounts for only 5–10% of all clinically presented cases (10
,74). Familial AD (FAD) can be classified as either early-onset or late-onset. The genes implicated in early-onset forms of the disease, which occur below 65 years of age are the APP gene located on chromosome 21q21; PSEN1 located on chromosome 14q24.3 and PSEN2 on chromosome 1q31–q42 (23,74,75). Mutations within the APP occur around the processing sites of the APP molecule resulting in increased production of the ß amyloid peptide (76). Most cases containing APP mutations have an age of onset between mid 40's and 50's but can be modified by the presence of the APOE genotype (57). Mutations within the APP gene appear to be family specific and do not occur within the majority of sporadic Alzheimer's cases. Missense mutations within the PSEN1 gene account for 18–50% of the early-onset autosomal dominant forms of AD (77). Mutations within the PSEN1 lead to a particularly aggressive form of the disease having an age of onset between 30 and 50 years, which is not influenced by the APOE genotype. However a polymorphism found within intron 8 of the PSEN1 gene was found to be associated with the development of the late-onset form of the disease (77,78). To date over 75 mutations have been found within the PSEN1 gene in families worldwide that are associated with the early-onset form of the disease (31). All mutations within PSEN1 increase production of the Aß42 (57,58). Mutations within PSEN2 have a variable age of onset (40–80 years), appear not to be influenced by APOE and result in increased ß amyloid peptide production (57).
The gene for APOE is located on chromosome 19q13.2 and certain polymorphisms are associated with the late-onset form of AD (>65 years) (23,79). The E3 allele is considered normal and occurs in 74% of the population; the E2 and E4 allele are less common, occurring in 10 and 16% of the population, respectively (10,79). The APOE gene does not cause Alzheimer's but acts as a marker altering individual risk based on possession of allelic combinations of the APOE4 allele (80). The highest risk is associated with the E4/E4 genotype. It has been proposed by Corder et al. (80), that each copy of the APOE4 allele reduces the age of onset by 7–9 years. An average age of onset of 85 years is given for individuals with no E4 alleles, 75 years for one allele and 68 years for individuals with two copies of the E4 allele (23,80).
Whereas genetic alterations in the previously mentioned four genes (Figure 7) are generally accepted as being implicated in causing FAD, other studies highlighting the potential role of other genes have come to light.
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Alpha 2 macroglobulin functions as a protease inhibitor and has been found in neuritic plaques. The gene is on chromosome 12p13.3 and variants of the gene are associated with the late-onset form of the disease (81
,82).
Genetic linkage analysis has identified a potential AD gene at or near the insulin degrading enzyme gene found on chromosome 10q23-25 which is involved in the cellular processing of insulin. At the current time further genetic analysis is being undertaken to determine a thorough assessment of this potential locus (83,84).
The monoamine oxidase A gene serves to regulate the metabolism of neuroactive and vasoactive amines within the central nervous system. Polymorphisms within this gene have also been implicated in the pathology of AD (85).
Myeloperoxidase (MPO) is an enzyme present in circulating monocytes and neutrophils and is part of the host's polymorphonuclear leukocyte defence system. MPO catalyses the production of the oxidant hypochlorous acid and is thought to contribute to Alzheimer's pathology through oxidation of either ß amyloid or APOE (86). These events could result in direct neuronal damage or promote insoluble amyloid complexes. It is interesting to note that the E4 isoform of APOE is the most readily oxidized and neurotoxic, conferring upon it a contributory role to increased risk for the disease (87). Microglia in normal brain tissue do not express MPO but it has been found to be expressed in the microglia of the senile plaques and neurons of the hippocampus and superior frontal gyrus within Alzheimer's brains (88). The gene for MPO resides on chromosome 17q23.1 and recently a polymorphism (G463A) has been associated with a 1.57-fold increased risk for AD in individuals carrying the MPO GG genotype (87). Novel polymorphisms found within the TAU gene have been associated with an increased risk for AD disease. A case–control study looking at the TAU IVSII+90G
A polymorphism showed a significant association between early age of onset in males and the possession of the A allele. This suggests that the risk factor for developing the disease may be modified as a result of both age and gender (89). A further case–control study examining the IVSII+34 GA polymorphism showed a 5-fold increased risk in individuals who carried both the APOE4 allele and were either heterozygous/homozygous for the G allele (GA, GG). This would suggest that the combined effect of both these alleles may have a significant outcome on Alzheimer's pathology (90). Hyperdiploidy of chromosome 17 would also result in over-expression of both the MPO and TAU gene and may be expected to contribute to the aetiology of the disease.
Mutations within genes such as APOE, CYP46A1 and ABCA1, which play a role in cholesterol and phospholipid metabolism, have been investigated and shown to have associations with AD. Recently a strong association between early-onset Alzheimer's and polymorphisms within the ABCA2 gene have been shown adding support for the role of these genes in Alzheimer's pathology (91).
Alpha-1-antichymotrypsin (ACT) is a protease inhibitor and is associated with astrocyte activity and the deposition of amyloid plaques. The G646T polymorphism within the promoter region of ACT has been shown to be associated with a higher risk for early-onset AD. This higher risk factor occurs independently of the APOE 4 genotype; however the rate of cognitive decline is elevated in those individuals homozygous for the ACT polymorphism and who also possess the APOE 4 genotype (92).
It has recently been suggested through the results of linkage analysis that the Ubiquilin 1 gene located at 9q22 could be a possible susceptibility gene for increased risk for AD (93). It plays an intriguing role in the degradation of proteins and has been shown to interact with both PSEN1 and PSEN2, promoting the accumulation of presenilin in vitro in cells over-expressing both Ubiquilin 1 and PSEN2 (94). Further investigations will need to be undertaken to further assess the role of Ubiquilin as a potential candidate risk gene for AD.
It is apparent that numerous families exist that possess the Alzheimer's phenotype but do not conform to the current pattern of known Alzheimer's genetics. This would indicate that there are as yet other loci waiting to be discovered that can act as genetic determinants for FAD.
Although much emphasis has been placed on the role of APOE as a risk factor, this is because of the current body of evidence that firmly establishes it in its role as a susceptibility gene relative to other known or unknown genetic risk factors. Other genes that have been reviewed are currently potential susceptibility genes but their status as definite risk factors can only be confirmed or refuted as additional evidence comes to light resulting from further investigation e.g. mutations within the progranulin gene on chromosome 17 have recently been found to play a role in dementia and may eventually be classified as a definite risk factor once these initial observations have been replicated (95).
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Genomic instability events |
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Aneuploidy
Damage to the genome could lead to altered gene dosage and gene expression as well as contribute to the risk of accelerated cell death in neuronal tissues. DNA damage events such as micronuclei formation, which are biomarkers of chromosome malsegregation and fragility, were found to be elevated in lymphocytes from individuals suffering from AD (96
). Micronuclei from Alzheimer's patients tended to be centromere positive relative to normal controls when examined with a centromeric probe, indicating whole chromosome loss rather than breakage, suggesting individual susceptibility to aneuploidy events possibly due to microtubule dysfunction (96,97). Fluorescent probes for chromosome 13 and 21 were hybridized to lymphocyte preparations from Alzheimer's patients and showed an elevation in aneuploidy for both chromosomes, but in particular for chromosome 21 (97,98). Further studies investigating fibroblasts from both spontaneous and familial Alzheimer's subjects carrying mutations in the genes PSEN1, PSEN2, and APP were found to have a 2-fold increase in the incidence of aneuploidy involving both chromosome 18 and 21 when compared to controls (99). It is possible that chromosome aneuploidy, particularly for chromosome 21, could be one of the mechanisms underlying both AD and the dementia that occurs in Down's syndrome patients (100).
Down's syndrome is diagnosed cytogenetically as being aneuploid for chromosome 21; individuals develop dementia that is histopathologically indistinguishable from AD during the third or fourth decade of life (101). Cells containing trisomy 21 are found in both Down's syndrome and Alzheimer's patients. Over-expression of the APP gene could lead to over-production of the Aß42 following cleavage by the secretase enzymes, which is causally related to plaque formation in both Down's syndrome and Alzheimer's brains (102). APP processing is also altered in terms of the ratio of Aß42 over the normal 40 amino acid ß amyloid peptide (Aß40), resulting in the increased production of the more amyloidogenic and neurotoxic form Aß42 (102). It has been proposed that individuals who are susceptible to AD may harbour significant numbers of trisomy 21 neural stem cells that with the passage of time, can result in brain segments having a Down's syndrome phenotype which may contribute to the aetiology of the disease (100). It is possible that like Down's syndrome this low-level mosaicism may have originated in utero as a result of age-related parental non-disjunction, or under dietary conditions such as low-folate status that has been shown to increase the rate of chromosome 17 and 21 aneuploidy (103). Alternatively, mosaicism may arise in those individuals who have a predisposition to aneuploidy events, which involve unequal mitotic segregation in aneuploid susceptible tissues within areas of the brain undergoing post-maturation neurogenesis. Such areas that may be affected include the dendate gyrus of the hippocampus that is involved in short-term memory and learning and is readily affected in the initial stages of Alzheimer's. These individuals are predisposed to develop the disease, but over a longer period of time due to the smaller population of mosaic cells that over-express genes that contribute to the symptoms of the disease such as APP and TAU. These hypotheses are biologically plausible however further evidence needs to be acquired to determine conclusively the potential roles of these mechanisms in Alzheimer's pathology. Mutated presenilin genes that contribute to early-onset FAD (104–106), produce proteins that have been found to be localized within the nuclear membrane, centrosomes and interphase kinetochores indicating a role in chromosome segregation and organization (99,106). Mutations within these genes that predispose to early-onset FAD (104–106), may alter the ability of the resultant proteins to lock on and release chromosomes to the nuclear membrane at the appropriate time during mitosis, hence leading to errors in chromosome segregation (106).
Aneuploid cells may be susceptible to programmed cell death directly leading to neurodegeneration by over-expressing presenilins and APP that increase the cells sensitivity to apoptotic stimuli (107). These cells having succumbed to apoptosis may then give rise to areas of inflammation, induced by the cerebral phagocytes microglia, that surround the neuritic plaques. Microglia induce astrocytes by expressing Interleukin-1. The astrocytes in turn express -chymotrypsin, which promotes further neurotoxic amyloid plaque formation (108,109).
Hyperphosphorylation of tau leads to a breakdown of the microtubule system, which may give rise to aneuploidy by causing defects in the mitotic spindle. Potentially an elevation in chromosome 17-aneuploidy could lead to an over-expression of the TAU gene resulting in abnormal production and accumulation of the protein initiating further neurofibrillary tangle formation. Additional chromosome 17-aneuploidy may occur as a result of elevated levels of oxidative stress that are also associated with AD (110).
Telomere shortening, oxidative stress, breakage fusion bridge cycles and gene amplification
Telomeres are hexanucleotide repeats that are located at the end of eukaryotic chromosomes and their dysfunction has been implicated in AD. They play an important role in maintaining genomic stability and preventing chromosomes from becoming tangled and protect the chromosomal ends from being recognized as a site of DNA damage (111,112). During DNA replication somatic cells lose telomeric repeats after every cell division undertaken, and therefore telomere length may serve as a marker of a cells replicative history. Telomerase and associated proteins prevent telomeres shortening in extreme proliferative cell types such as cancer cell lines and stem cells. Telomerase is also present in other cells such as lymphocytes but at levels that cannot prevent shortening of the telomeres leading to replicative senescence (111,113).
It has been shown that telomere shortening is associated with an elevated incidence of certain cancers (head, neck, lungs, epithelial cancers such as prostate) and is exacerbated by other risk factors such as ageing and smoking (114). Other studies have shown that individuals exhibiting accelerated telomere shortening die 4–5 years earlier and have higher incidences of heart disease compared to age and gender matched controls (115). Telomere shortening results in a decrease in lymphocyte proliferation activity and has been shown to impair the immune system in AD. Telomere shortening is also associated with T cell clonal expansion involving immune responses to auto antigens in Alzheimer's patients. Panossian et al. (112) found significant telomere shortening in peripheral blood mononuclear cells from Alzheimer's patients compared to controls. T cell telomere length correlated with deficits in cognitive function, as assessed by the mini mental state examination, which determines cognitive decline (112). Telomeres have also been found to be significantly shorter in leukocytes of individuals suffering from vascular dementia compared to age and gender matched controls (116). Genomic instability resulting from telomere loss may lead to gene over-expression as a result of gene amplification, through the repeated breakage and fusion of chromosomes that occur through the breakage/fusion/bridge cycle (BFB) (117–119).
The cycle can be initiated by telomere end fusions with short telomeres, which fuse to form a dicentric chromosome. If the fusion occurs between homologous chromosomes the dicentric chromosome will contain two copies of a gene positioned between two centromeres. These centromeres are drawn to opposite poles during anaphase and break unevenly as cytokinesis occurs. This results in a chromosome containing two copies of a gene and a fragment that has lost its initial gene copy. These multiple gene containing chromatids may further fuse during interphase to form another dicentric chromosome increasing its gene complement which is then replicated during nuclear division resulting in further amplification following the next BFB cycle (120,121) (Figure 8).
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It has been shown that smaller chromosomes such as chromosome 17 and 21 have shorter telomeres than their larger genomic counterparts (122
). It may be possible that these smaller chromosomes may be more susceptible to undergo BFB cycles (123), resulting in over-expression of Alzheimer's-related genes such as TAU and APP. This hypothesis has biological plausibility but further evidence is required in order to determine conclusively its role in Alzheimer's pathology.
Differences in the telomere length of human chromosomes may be a contributory factor relating to a higher incidence of specific chromosome aneuploidy (124). Telomeres are implicated in the positioning of chromosomes by forming a point of attachment to the nuclear membrane within the interphase nucleus and in chromatid segregation during mitosis. Smaller chromosomes with shorter telomeres such as chromosomes 17 and 21 may be more susceptible to aneuploidy events as a result of mitotic non-disjunction events or faulty interphase chromosomal positioning. It is also possible that BFB cycle events involving dicentric chromosomes containing homologous chromosomes may result in aneuploidy through the formation of chromosomal end-to-end fusions resulting in subsequent gene amplification (122,125). It has also been shown that telomere shortening is accelerated under conditions of oxidative stress (116,126). Some of the measurable genome instability events are seen in Figure 9.
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Oxidative stress is now accepted as playing a key role in the pathology of AD (127
–129), and is the result of an imbalance between elevated free radical production and a decrease in either free radical scavenging or the mechanisms used to repair oxidized macromolecules. This leads invariably to cellular dysfunction and eventual cell death.
Free radicals such as the hydroxyl ion, hydrogen peroxide and peroxynitrite have been shown to induce cytotoxicity by interacting with and damaging major components of the cell through oxidation of lipids and nucleic acids including the nucleus, membrane and cytoplasmic proteins, mitochondrial DNA, as well as, being involved with neurotoxic metal complexes, thereby compromising their cellular function. Neurons appear to be particularly vulnerable to the damaging effects of free radicals, they are known to have low levels of natural antioxidants such as glutathione which would aid in protecting the neuron (43), as well as requiring an increased oxygen demand to maintain brain metabolism (130). The elevated oxygen environment is ideal as it can be used as a substrate in reactions with compounds that are primary producers of free radicals e.g. ß amyloid.
APOE may be beneficial by reducing neuronal death caused by ß amyloid and hydrogen peroxide by acting as an antioxidant (131). Of the three allelic states the E2 isoform is the most effective antioxidant with the E4 allele (associated with late-onset AD) being the least effective in its protective capacity (132). However it has also been shown that the APOE4 isoform is the most sensitive to free radical attack compared with the more resilient E2 isoform and that peroxidation levels in the brains of AD patients that possessed the E4 allele are elevated compared to those individuals possessing either the E2 or E3 allele (133).
It has recently been shown using a modified version of the comet assay that there is a 2-fold increase in levels of DNA damage and oxidized DNA bases (pyrimidines and purines) in leukocytes of individuals with mild cognitive impairment (MCI) and AD when compared to matched-controls (134). MCI individuals display the initial signs of cognitive decline (memory loss) but not to the extent that they cannot pursue or participate in everyday activities. This suggests that oxidative damage occurs at the early stages of AD, as MCI patient's progress to Alzheimer's with an estimated probability of 50% within 4 years or 12% per year (134,135), suggesting that MCI represents the early stages of the disease and that oxidative stress directly contributes to disease pathology.
A further marker of oxidative stress 8-Hydroxy-2-deoxyguanosine (8-OHdG) is elevated in both peripheral tissues of MCI and AD (129,134,136). It has also been shown that oxidative stress is clear within the post mortem Alzheimer's brain where an increase in mitochondrial and nuclear oxidative damage is evident (44,135). Interestingly it appears that an increase in both 8-OHdG and 3-Nitrotyrosine within the Alzheimer's brain occur prior to the development of both tangles and plaques indicating that oxidative stress may be one of the initiating factors leading to further genomic instability (44,136,137).
The genome instability model of AD outlined in Figure 10 allows for the possibility that certain disease states may have already been pre-determined in utero. Aneuploidy prone individuals (either as a result of individual genome or micronutrient deficiency e.g. folate) can give rise to low levels of mosaicism for individual chromosomes such as chromosome 17 and 21. Gene amplification may occur as a result of telomere end fusions (Figure 8). Aneuploidy may result in additional copies of both APP and TAU leading to the altered homeostasis of both these proteins. The Alzheimer's phenotype becomes apparent in stem cells, which differentiate into neural stem cells with abnormal APP and tau levels, which with the passage of time influence the initial stages of neurodegeneration. These stem cells could also differentiate into non-neural somatic cells (e.g. buccal or fibroblast which having an elevated tau and APP level) and could be used to study genomic instability events such as elevated micronuclei and aneuploidy.
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Dietary and nutrigenetic factors that affect Alzheimer disease risk |
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 TopIntroductionClinical diagnosisTau and neurofibrillary tanglesß amyloid and neuritic...Amyloid cascade hypothesis:...Genetics of ADGenomic instability events Dietary and nutrigenetic factors... ConclusionReferences |
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B vitamins
Many case–control studies have shown that Alzheimer's patients have been found to be deficient in certain micronutrients such as folate, vitamin B12 but have elevated levels of the sulphur based amino acid homocysteine (138
,139). These factors are associated with increased micronucleus formation and the alteration of methylation patterns that could modify gene expression (120,140,141). Folate and vitamin B12 play an important role in DNA metabolism. Folate is the methyl donor for the conversion of dTMP from dUMP, which is required for DNA synthesis and repair. Under conditions of low folate, dUMP accumulates producing DNA strand breaks and micronucleus formation as a result of uracil misincorporation into DNA in place of thymine (120,142). Both folate and vitamin B12 are required in the synthesis of methionine through the remethylation of homocysteine, and in the synthesis of S-adenosyl methionine (SAM), which is involved in maintaining genomic methylation patterns that determine gene expression (143). Folate deficiency reduces SAM resulting in the depletion of cytosine methylation in DNA, and thereby elevating homocysteine levels. Additionally folate deficiency has been shown to increase trisomy of chromosome 17 and 21, which code for the TAU and APP genes, respectively (103).
Hyperhomocysteinemia has been shown to be a strong independent risk factor for AD in a number of epidemiological studies (144–148). It appears that nervous tissue may be extremely sensitive to excessive homocysteine as it promotes excitotoxicity and damages neuronal DNA giving rise to apoptosis (149). Studies have also shown a strong correlation between a reduction in hippocampal width, which is associated with short-term memory loss, and concentrations of plasma homocysteine (150). Recently MRI measurements have shown that an inverse relationship exists between plasma homocysteine and cortical and hippocampal volume (151). The above findings have been interpreted as involving neuronal damage within the hippocampal regions leading to memory loss, which is characteristic of AD. Elevated homocysteine has also been implicated as playing a role in an iron dysregulation/oxidative stress cycle that is thought to be central to the pathogenesis of the disease (152).
Homocysteine levels are usually maintained within physiologically correct limits by remethylation to methionine in a reaction involving folate and vitamin B12 (Figure 11). Homocysteine can also be converted to cystathionine by the involvement of the enzyme cystathionine ß-synthase, resulting in increased levels of glutathione which is a natural anti oxidant (153).