Prioritization and functional analysis of
1. Introduction
As Alois Alzheimer himself first observed, the brain of an individual affected by Alzheimer's disease (AD) shows aggregations of the peptide beta-amyloid (Aβ) and tau proteins, which form characteristic plaques and neurofibrillary tangles respectively [1].
Since Aβ is known to participate in many normal body functions, its precipitation into plaques, often referred to as ‘amyloid cascade’, has been for a long time the only recognized, yet unexplained mechanism of AD pathogenesis [2]. In time, however, diverse phenomena, such as oxidative stress, aberrant inflammations, impaired energy metabolism and more have been gradually discovered to contribute to the cascade [3].
The observation that Aβ aggregation in plaques is an age-dependent phenomenon, whereas Aβ production is not, suggested that some other age-dependent mechanism must play a role in transforming Aβ into a neurotoxic element. The fact that some metals, such as copper and zinc, are known to modulate glutamatergic neurotransmission [4] led researchers to hypothesize that late-age abnormalities in the homeostasis of one or more transition metals may play a role in the amyloid cascade.
Moreover, much evidence gathered on AD depicts an improperly functioning ceruloplasmin, an enzyme synthesized by the liver, which controls iron oxidization state. There is also evidence that a functional failure of systemic ceruloplasmin may be behind the iron-related redox processes that produce oxidative stress in the AD brain [5, 6]. Ceruloplasmin is the ‘crosstalk’ factor linking copper to iron metabolism, thus its failure is very likely to be a major actor in the dysfunctional metal metabolism affecting AD individuals. Aβ may gain toxicity upon some interaction with copper, in a process involving ceruloplasmin, even though the molecular mechanism still remains elusive.
This notion was further supported by the fact that the Amyloid precursor protein (APP) was discovered to possess selective copper binding sites, which mediate redox activities causing precipitation of Aβ even at low concentrations [7]. Aβ itself has been reported to possess selective high- and low-affinity metal-binding sites which, in normal conditions, bind equimolar amounts of either copper or zinc but, in conditions of acidosis, see zinc completely displaced by copper [8]. Thus, hyper-metallation was suggested to be the mechanism that gives Aβ its redox properties, triggering redox cycles through production of H2O2 that lead to self-oxidation of Aβ, formation of oligomers with diverse grades of complexity and finally to Aβ precipitation into plaques (Figure 1) [4, 9].
Diverse animal studies support the toxic role of copper in AD pathogenesis.
White et al. [10] showed that the copper contents of both liver and cerebral cortex in
Sparks and Schreurs [11] demonstrated that adding 0.12 ppm (0.12 mg/L) of copper to water given to a cholesterol-fed rabbit AD-model resulted in significantly enhanced cognitive waning and also exacerbated amyloid plaque deposition. This finding led to serious concerns in some Government Environmental Agencies about the content of copper in drinking water delivered to households via copper pipes. It must be kept in mind that cholesterol, though vital for neuronal transmission, synaptic plasticity and cell function, is also a well-established risk factor for atherosclerosis and AD [12]. Cholesterol to 7-hydroxy cholesterol oxidation, caused by Aβ, is extremely toxic for neurons [13]. Cp levels measured using o-dianisidine dihydrochloride as a substrate in the plasma of cholesterol-fed rabbit model after adding copper to drinking water, suggest an increase, although the change did not reach statistical significance. This suggests that a Non-Cp copper increase is a vehicle of copper within the brain.
In a different investigation [14], Sparks reconfirmed this earlier finding, reporting that other animal models, like spontaneously hyper-cholesterolemic Watanabe rabbits, cholesterol-fed beagles and rabbits, and
In another study [15], Lu at al. confirmed the findings of Sparks and colleagues’. The authors demonstrated that Kunming strain mice fed with a high-cholesterol diet and distilled water containing 0.21 ppm copper exhibited significantly increased level of
Moreover, it has been reported [16] that, in Sprague-Dawley rats, which underwent bilateral common carotid artery occlusion (2VO) and were administered with 250 ppm copper containing water for 3 months, chronic copper toxicity exacerbated memory impairment induced by 2VO coupled with an augmented expression of brain AβPP and β-site AβPP-cleaving enzyme 1 (BACE1) at both mRNA and protein levels. However, these copper-aggravated changes were ameliorated after copper was withdrawn from the drinking water.
As a whole, these experimental animal models demonstrated the toxicity mediated by copper in the AD cascade, showing that increased level of copper ingested with drinking water, or more generally through the diet, affects AD neuropathology.
All this evidence has eventually led to the proposal of the so called Metal Hypothesis of AD (Bush et al. 2008), which is based on the concept that it is the interaction of Aβ with specific metals, especially copper, that actually drives the amyloid cascade and AD pathogenesis.
One question remained: how does copper actually reach the brain? In fact, we normally ingest copper through the diet - via food, drinking water, beverage, supplements - and copper status in the body is regulated by the balance between duodenal absorption (intestine) and biliary excretion (liver). After crossing the intestinal lumen, copper is transported via portal circulation to the liver, where it is partly stored and partly redistributed to other organs. In the hepatocyte, copper is incorporated into ceruloplasmin, whose dimensions don’t allow an easy crossing of the blood-brain-barrier (BBB).
An answer to the question came with the discovery that, although the vast majority of human copper circulates tightly bound to ceruloplasmin [17, 18], a faulty copper metabolism leads to the creation of a small pool of copper that goes into circulation loosely bound to and constantly exchanged among albumin, α2 macroglobulin, peptides, amino acids and other low-molecular-weight compounds. Due to the loose character of the bindings, this portion is normally referred to as Non Ceruloplasmin copper (Non–Cp copper). The key difference between bound (to ceruloplasmin) and Non-Cp copper lies in the fact that the low-molecular-weight compounds can easily cross the BBB [18], thus carrying Non-Cp copper into the brain. There, copper can enter cycles of Haber-Weiss or Fenton reactions producing ⋅OH, against which our body has no defenses [19], and generate pleiotropic effects on the amyloid cascade [3].
The metal hypothesis has also gained support from consistent reports of enhanced concentrations of labile copper in areas of the brain that are considered critical for AD [20].
There is by now a solid body of literature reporting
Also results of a recently completed Phase II clinical trial, based on using metal attenuating complexing compounds or Zinc therapy [34-37], appear to support the notion of a copper dysfunction in AD. The available evidence has now reached such a quantitative and qualitative level that the notion of a copper-related phenotype in AD has now started to be accepted [23]. This is a very important step, since many translational hypotheses may develop from this notion, in terms of both diagnostic and prognostic tools, with important repercussions in terms of preventive and therapeutic approaches. However, most of the literature dealing with the relationship between copper and AD focuses on local copper abnormal distribution, especially in those specific areas of the brain that are considered critical for the disease. Recently, this vision has started to appear limited. There is now a bulk of evidence suggesting that all modifications should be viewed in a wider framework of systemic, rather than local, metal dishomeostasis. This concept can be better understood looking at recent studies of the link between the status of serum ceruloplasmin and AD clinical signs and/or Aβ markers in the CSF [23]. Torsdottir et al. [38] reported a decrease in ceruloplasmin activity in AD patients. Lower levels of circulating ceruloplasmin in AD patients with different CSF markers of AD were reported by Brewer’s [39], Arnal’s [40] and Kessler’s [41] groups. In 2008, our laboratory demonstrated a consistent and measurable increase of apo-ceruloplasmin (a defective form of ceruloplasmin, lacking copper and its ferroxidase activity) in the serum and CSF of AD patients [42, 43].
Since both WD and the early-onset form of AD are known to have a genetic origin determining the hereditability of the disease, researchers have embarked in a wide range of studies in the attempt to find genes that cause the late-onset AD or at least contribute to it via damaging phenomena, such as oxidative stress, inflammation, apoptosis or an increased expression of Aβ. In order to encompass as much as possible of the huge genome world, researchers have also embarked in so-called large-scale genome-wide association studies (GWAS). These studies search for DNA sequence variations that appear more common in individuals with a certain disease than in individuals without that disease. GWAS typically analyze a multitude of single gene variations, generally called single-nucleotide polymorphisms (SNPs), and verify their association, if any, with the traits of a disease.
2. AD and APOE
So far, no specific gene has been found that can be reliably considered a cause of AD. Even genes, whose mutations have been found responsible for early-onset AD, appear to have a minor, or at least not a pivotal role in the late-onset form. However, numerous risk factors have been identified in the last few years. Historically, the first one to be established is the inheritance of the ε4 allele of the apolipoprotein E (
Individuals inherit two copies of the
It must be emphasized, though, that we are dealing here with statistical risk: in fact, not all individuals who have one or two ε4 develop AD and AD occurs also in people who have no ε4. Thus,
3. Other genes
The fact that
In AD, GWAS have consistently shown that the effect size and the strength of association of
Another approach is selecting a candidate gene on the basis of hypotheses regarding the disease and then analyze all sequence changes of that gene. Direct sequencing has proven an effective way to discover rare variants with large effect sizes. Recently, studies using next-generation sequencing have led to the identification of rare frequency coding variants in
3.1. ATP7A and ATP7B
Two serious disorders are today recognized to be due to a dyshomeostasis in copper metabolism: Menkes disease (MD) and Wilson’s disease (WD). Both are caused by a mutation of one gene:
The
Technically, mutations of the two genes have somewhat opposite effects: in MD, the dysfunctional
We know that in WD huge amounts of Non-Cp copper enter the brain through the BBB, where labile copper accumulates and leads to neurodegeneration. WD is considered the hallmark of copper toxicosis.
Since increased copper levels characterizing WD are shared in a smaller scale by AD patients, the
Our laboratory has pursued the link between copper and AD pathogenesis on the assumption that the excessive Non-Cp copper production in the body is actually due to a faulty ATP7B causing a flaw in the incorporation of copper into nascent ceruloplasmin in the liver [56]. On this basis, we have embarked in an extensive study of the
Unfortunately, analysis of the
The first significant information on the structure of the ATP7B protein was gained from a homology modeling study [65]. Some more light was later shed by nuclear magnetic resonance (NMR) spectroscopy studies [66-70]. Recently, significant progress in the comprehension of ATP7B structural organization has come from the solution of the crystal structure of the bacterial copper ATPase LCopA [71]. The LCopA protein model has been employed as a template to analyze ATP7B core domain on the basis of its sequence homology to build interpretations of WD mutations [64].
We know that mutations leading to a complete abolition of ATP7B function, chiefly early stop mutations and mutations in regions of the gene that have a high functional importance, lead to an early and predominantly hepatic dysfunction. Conversely, point mutations in regions that are functionally less important are associated with a later onset and predominantly neurological or psychiatric dysfunctions [17].
An effective strategy to characterize genetic compositions, which has become popular in recent years, is the so-called
Our laboratory has used a new in-silico approach based on amino acid sequence, utilizing four among the most used bioinformatics tools (i.e. Polyphen- 2, SIFT, Panther, and PhD-SNPs). We have applied this approach to non-synonymous SNP (nsSNPs) detected in the
3.2. rs7323774 and rs2147363
In pursuit of the identification of regions in the
In one study of 399 AD patients and 303 healthy elderly controls, we focused our attention on a set of four SNPs that had been reported to be informative of the
We first stratified the AD and control groups into three ‘classes’ according to their Non-Cp copper levels: ‘low Non-Cp copper’ (<1 µmol/L), ‘medium Non-Cp copper’ (≥1, <1.6 µmol/L) and ‘high Non-Cp copper’ (≥1.6 µmol/L) (Figure 2). Results showed antithetic distributions for patients and controls: the ‘low Non-Cp copper’ class accounted for 27% of AD cases vs. 61% of controls; the ‘medium Non-Cp copper’ for 11% of AD vs. 10% of controls and the ‘high Non-Cp copper’ for 62% of AD versus 29% of controls. The serum copper profile was then analyzed in relation to the selected gene variants in the sole ‘high Non-Cp copper’ class (patients with AD, n = 109; controls, n = 53).
The main result of this study was that individuals who are GG homozygous for
In another study of 286 AD patients and 283 controls we focused on the rs2147363 and rs7334118 variants of the
We analyzed the genetic association between SNPs and AD risk. Our study revealed a significant association between rs2147363 and AD. When data were adjusted for confounding variables (i.e., age, gender, and
In order to verify whether rs2147363 has any functional variants in LD, we also analyzed the SNPs that showed a complete LD (D’ = 1) with this
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Intronic enhancer | intronic | - | - |
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Intronic enhancer | intronic | - | - |
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Intronic enhancer | intronic | - | - |
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Intronic enhancer | intronic | - | - |
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Intronic with unknown function | intronic | - | - |
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Intronic with unknown function | intronic | - | - |
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Intronic with unknown function | intronic | - | - |
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Downstream with unknown function | 3-UTR | - | - |
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Intronic with unknown function | intronic | - | - |
To predict the functional impact of the non-synonymous SNP (rs7334118), we used two different bioinformatics tools: SIFT and Polyphen2. The application of both the SIFT algorithm using orthologous sequences and the Polyphen2 tool highlighted that this coding variant may have an adverse effect on the ATP7B protein.
To verify the hypothesis that genetic association of rs2147363 is due to LD with the rs7334118 SNP, 176 AD subjects and 169 healthy controls among the study population were genotyped for the SNP rs7334118. Two AD patients were carriers of the rs7334118G allele, whereas no healthy individual with this
To test whether the intronic region, in which rs2147363 is located, plays a role in the regulation of the
We used the HapMap database to identify functional coding variants, in which only few rare variants are present. Using other genomic databases, such as the database of 1,000 Genomes Project, may deliver new candidate variants that could be tested to explain our intronic association. Conversely, in-silico analyses on rs2147363 revealed that this intronic SNP can have a regulatory role on ATP7B function. In particular, the GERP analysis highlighted that the intronic region around rs2147363 resulted significantly conserved, and the is-rSNP algorithm significantly predicted the presence of two TF-binding sites in the rs2147363 region. These independent findings suggested that this genomic region has a regulatory function, and consequently rs2147363 can be associated with clinical phenotypes related to ATP7B dysfunction.
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G | -7.84 |
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G | 0.889 |
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C | -1.02 |
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C | 1.93 |
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T | -0.596 |
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T | -3.02 |
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G | -3.44 |
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T | -7.19 |
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Specifically, Zfp423 and PLAG1 have been predicted to have a binding-site in the genetic region of rs2147363. Both these TFs are involved in complex metabolic processes. For example, Zfp423 has been reported to regulate neural and adipocyte development [78], whereas
3.3. K832R (rs1061472) and R952K (rs732774)
In a recent study, we focused our attention on two functional changes in the
K832R corresponds to a non synonymous amino acid substitution and the corresponding AAG>AGG mutation in exon 10 specifies this amino acid change in the A-domain, within the ATP binding domain region of the protein. R952K (AGA>AAA) corresponds to a non-synonymous substitution in the loop between Tm 5-Tm6 of the protein (Figure 3). Our aim was to verify whether and in what way these amino acid changes have a disturbing effect on the function of the ATP7B protein in terms of metal binding properties or ATP hydrolysis, which can eventually result in copper dyshomeostasis.
We recruited 251 AD patients and 201 controls. As reported in the original article [76], for the K832R substitution, the minor allele frequency (MAF) resulted 40% in Italian Tuscans, 45% in Utah residents with Northern and Western European ancestry, while 42% in our controls. For the R952K substitution, MAF was 39% in Italian Tuscans, 44% in Utah residents and 43% in controls. The LD analysis revealed an association between K832R and R952K substitutions in both AD patients (D’ = 0.79) and controls (D’ = 0.81). A high LD between K832R and R952K was confirmed also in all HapMap populations.
Allele frequency distributions of both
The χ2 test indicated that two distributions differed (p = 0.006) and the association between the risk allele K952 and AD was maintained after checking for age and gender as possible confounders in a logistic regression model. In summary, patients with the
We also performed a haplotype association analysis for the two SNPs in order to investigate their combined effect on AD risk. The most common haplotype was R832/K952, which contained a risk allele at each SNP locus. It was distributed as follows: 60.2% in AD patients and 53.2% in controls (X2 = 4.85; p = 0.028).
The second more frequent haplotype was K832/R952, which contained no risk alleles and its frequencies differed between the two cohorts, being 28.8% in patients and 37.3% in controls (X2 = 7.21; p = 0.007).
A logistic regression model was used to check these associations when taking into account age as a possible confounder. The model confirmed the association (p = 0.018) and revealed that the haplotype K832/R952 confers some protection against AD [adjusted OR= 0.68 (0.49–0.93)]. Thus, the haplotype association analysis revealed that the presence of alleles with normal function, i.e., K832 and R952, is protective, even though the haplotype lies in a gene with significant disease-risk.
It is important to notice that the SNPs of
4. Conclusions
The form of AD that has been the subject of this chapter accounts for about 95% of all AD cases and appears rather late in life, normally after 60. For this reason it is called late-onset Alzheimer's disease (LOAD), a term that well differentiates it from the inherited form, familial AD, which is called early-onset AD because it develops much before 60 and accounts for the remainder 5%. In opposition to familial AD, LOAD was initially called "sporadic" because early researchers saw no link between the disease and hereditary factors and assumed that the appearance of the disease was occasional and totally casual.
As we have seen above, this appeared untrue later, when researchers discovered the role of several polymorphisms of the
Due to the lack of a secure culprit, the role of genetic mutations in LOAD is often understated. In an attempt to compensate, this chapter has described some of the most meaningful evidence constituting the genetic background believed to provide a combined contribution to susceptibility (to become sick), which remains a statistical entity. It must be kept in mind that not only genetic heterogeneity contributes to susceptibility but also other factors, such as
The chapter has also described GWAS, which have linked a substantial number of genes to the pathogenesis of AD. It is important to notice that each gene, when taken alone, accounts only for a small percentage of the disease incidence and often lacks clinical significance: the above mentioned
However, all the genetic evidence presented here should be regarded as the framework in which systemic metal unbalances develop. For this reason, we have described in a consequential but also factual fashion the role of systemic copper in the toxic processes leading to AD, which we believe to be among the most important phenomena in AD pathogenesis.
We conclude with the introduction of an important, and somewhat innovative, notion concerning copper in AD. We have shown how multiple variants of the
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