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Differential Expression of Ribosomal Genes in Brain and Blood of Alzheimer’s Disease Patients: Original Research Lucas Rasmussen1, Roger W. de Labio2, Gustavo A. Viani2, Elizabeth Chen3, João Villares, Paulo-Henrique Bertolucci3, Thais S. Minett4, Gustavo Turecki5, Danielle Cécyre5, Sandra A. Drigo6, Marilia C. Smith3 and Spencer L.M. Payão1,2,* 1
Sagrado Coração University (Universidade Sagrado Coração), Bauru, São Paulo, Brazil; 2Marília School of Medicine (Faculdade de Medicina de Marília), Marília São Paulo, Brazil; 3Federal University of São Paulo (Universidade Federal de São Paulo), São Paulo, Brazil; 4University of Cambridge, Cambridge, United Kingdom; 5Bell Canada Brain Bank,Douglas Mental Health University Institute,Montreal Quebec, Canada; 6NeoGene Laboratory, São Paulo State University School of Medicine (Faculdade de Medicina da UNESP), São Paulo, Brazil
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Abstract: Changes in rRNA and rDNA expression have been associated with cellular and organism aging and have been linked to Alzheimer’s disease (AD) pathogenesis. In this study, we investigated the mRNA expression of ribosomal genes (28S/18S) and β-amyloid precursor protein (APP) in different post mortem brain tissue regions (the entorhinal and auditory cortices and the hippocampus) of AD patients and elderly control subjects and also evaluated the extent of expression in peripheral blood from young, healthy, elderly, and Alzheimer’s disease patients in order to investigate whether these individuals experienced the effects of aging. The comparative threshold cycle (CT) method via Real Time Polymerase Chain Reaction and the Polymerase Chain ReactionRestriction Fragment Length Polymorphism (PCR-RFLP) were used to analyze gene expression and the Apolipoprotein E (APOE) genotype, respectively. When the brain areas were analyzed collectively, we observed a significant decrease in APP expression and a significant increase in levels of mRNA of 18S and 28S in Alzheimer’s disease patients compared to healthy elderly individuals. Furthermore, there was a significant upregulation of 28SrRNA in the entorhinal cortex and hippocampus, but not in the auditory cortex of patients with AD. On the other hand, tests of blood samples verified a decreased expression of 28S rRNA in patients with AD. These results support the hypothesis that changes in rRNA are present in AD patients, are tissue-specific, and seem to occur independently and differently in each tissue. However, the next challenge is to discover the mechanisms responsible for the differences in expression observed in the blood and the brain in both healthy elderly individuals and Alzheimer’s disease patients, as well as the impact of these genes on AD pathogenesis.
Keywords: Alzheimer’s Disease, Brain, Blood, Ribosomal Genes, 18S, 28S. INTRODUCTION The prevalence of neurodegenerative disorders increases with age, and molecular processes associated with brain aging contribute to cognitive decline [1, 2]. Alzheimer's disease is the most common form of age-related dementia. This condition is characterized by chronic neurodegeneration that leads to progressive memory deficits, cognitive impairment, personality changes, and behavioral and emotional disturbances [3, 4]. While senile plaques are normal by-products of senescence, the accumulation of aggregated β-amyloids and neurofibrillary tangles are have been linked to Alzheimer’s disease and its associated symptoms [5, 6]. In the brain, the abundance of Aβ peptides that are derived from the APP are usually located in the cortex and hip*Address correspondence to this author at the Genetics Laboratory, Hematology Center, Marilia Schoolf of Medicine (FAMEMA) Rua Lourival Freire, 240, Bairro Fragata, CEP 17519-050, Marília, São Paulo, Brazil; Tel: +55 14 34021856; Fax: +55 14 34330148; E-mail:
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pocampus and are correlated with neuronal death and synaptic dysfunction [7]. In addition, Aβ peptide deposits result in neuroinflammation and neurovascular inflammation, which contribute to neurodegeneration in Alzheimer's disease [8]. The events contributing to AD development are numerous and complex. Many genes have been described as being deregulated in AD, and several of these genes have been linked to cognitive impairment [9]. There are various potential risks, but the presence of Apolipoprotein E4 (ApoE4) polymorphism is the most common genetic risk factor in the development of AD [10, 11]. According to Tokuda, et al. [12], ApoE4 is linked to the mechanism of AD pathogenesis because it has been associated with a greater Aβ burden than the other isoforms, ApoE4 binds to Aβ with lower affinity, which may result in less efficient clearing of Aβ through the cell surface [12, 13]. Leduc, et al. [14] reported that APOE4 is also associated with greater neuronal inflammation and less efficient neuronal repair. © 2015 Bentham Science Publishers
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Aβ is released by the APP through sequential cleavages by β-secretase and y-secretase. This important event has been the focus of much research. However, previous work from our group has also suggested that ribosomal genes may play a role in the development of AD [15-18]. Ribosomal genes codify the 18S, 5.8S and 28S ribosomal RNA (rRNA). The expression of repeated nucleolar rRNA genes (rDNA) initiates ribosomal biogenesis and is a subject of complex regulation. Several studies affirm that the ribosome is the nexus for all cellular protein translation and that a reduction in ribosomal activity may be a major contributor to Alzheimer’s disease pathology (AD) [19]. Kalita, et al. [20] and Rieker, et al. [21] showed that, in rat and mouse neurons, blocking nucleolar transcription induces neurodegeneration, including neuronal death. Taken together, these genes (APP, Ribosomal RNA, and ApoE) are assumed to be responsible for approximately 50% of the genetic background of the disease; the APP process, which itself is influenced by ApoE and associated with 18S/28S alterations that compromise ribosomal biogenesis, contributes to development of AD. The literature is not clear on the pathological mechanisms of AD; however, changes in gene expression in the brain of patients with AD may be associated with the development of neurological disease. Therefore, the purpose of this study was to genotype ApoE and to investigate the expression of ribosomal genes (28S/18S) and APP in the hippocampus, the entorhinal cortex, and the auditory cortex, as well as in the peripheral blood of AD patients when compared to samples from controls. MATERIALS AND METHODS Patient Characteristics and Samples This study includes brain and blood samples from both individuals with Alzheimer’s disease and healthy individuals whose samples were used as the control. It is important to emphasize that the brain and blood samples used did not come from the same individual. The great difficulty in obtaining brain samples from individuals with Alzheimer's and from healthy controls restricts the collection of both samples in the same subjects. The brain and blood samples, autopsy consents, neuropathological assessments, and other research, including the genetic studies, were all obtained from in accordance with the local Research Ethics Committee. The Institutional Research Ethics Committee approved this study, and all subjects or their legal representatives each signed an informed consent according to the Declaration of Helsinki.
Rasmussen et al.
auditory cortex (49 total samples: 25 from healthy elderly individuals and 24 from AD patients) and the hippocampus (50 total samples: 25 from healthy elderly individuals and 25 from AD patients). Neither sex nor age differed among the studied groups (p>0.05). All the characteristics of the samples are described in Table 1. Thirty-seven brain tissue samples were obtained in collaboration with the Douglas Hospital Research Center Brain Bank (Montreal, Canada), and fifteen were obtained in collaboration the Federal University of São Paulo, (São Paulo, Brazil). All of the brain tissue samples (with or without AD) were processed in the same way using identical sampling protocols. The post-mortem interval between time of death and tissue freezing ranged from 7 to 37.5 hours. To be included, patients had to be over 60 years of age and presenting symptoms consistent with the criteria for probable AD from the National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) and of the IV Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) [22]. Neuropathological characterization of AD was determined according to plaque and tangle formation, neuronal loss, cerebral atrophy, and cognitive impairment. Blood Samples Both genotyping and gene expression in peripheral blood lymphocytes were studied in 82 healthy young control patients, 83 healthy elderly control patients and, and 82 subjects with AD. The three subject groups had similar ethnic origins: 95% were of European descent, 2.5%, were of Japanese descent, and 2.5% were multi-ethnic. There was no difference in age or sex between the AD group and the healthy elderly groups (p>0.05) (Table 1). The subjects were evaluated using the Mini-Mental State Examination (MMSE) and the Katz index [23, 24]. AD patients were selected according to the National Institute of Neurological and Communicative Disorders and Stroke Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) criteria and the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) for probable AD [22]. The MMSE, the Katz index, and the Clinical Dementia Rating (CDR) were also evaluated by Morris, et al. [25]. All of the patients were recruited from the Department of Neurology at the Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil. Our methods and results follow the MIQE checklist. DNA Extraction and ApoE Genotyping
Human Brain Samples We analyzed DNA and RNA samples from 52 individuals, 26 AD patients (mean age ± standard deviation (SD) 82.14 ± 6.43 years; sex 14♀/12♂) and 26 healthy elderly (mean age ± SD 75.45 ± 9.32 years; sex 14♀/12♂). DNA samples were analyzed from all individuals. In addition, 150 RNA samples were obtained from three different regions of the brain from all individuals. These regions included the entorhinal cortex (51 total samples 26 from healthy elderly individuals and 25 from AD patients), the
Genomic DNA from the brain and blood samples was extracted using the QIAamp Tissue Kit and QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. ApoE genotypes (rs429358, rs7412) were determined using PCR-RFLP according to our previously established protocols [26]. RNA Extraction and cDNA Synthesis Total RNA from the brain tissue was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Germany), ac-
rRNA in Brain and Blood of Alzheimer's Patients
Table 1.
Current Alzheimer Research, 2015, Vol. 12, No. 10 3
Characterization of brain samples from all subjects.
Tissue
Group
n
Brain Region
N
Mean Age ± SD
Gender ♀/♂
Healthy elderly
26
Entorhinal Cortices Auditory Cortices Hippocampus
26 25 25
75.45 (± 9.32)
17♀/9♂
Alzheimer Disease
26
Entorhinal Cortices Auditory Cortices Hippocampus
25 24 25
82.14 (±6.43)
14♀/12♂
Healthy young Healthy elderly Alzheimer Disease
82 83 82
-----------------------------------------------------------------
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19 (± 1.78) 71.73 (± 8.95) 74.5 (± 8.52)
28♀/54♂
Brain
Blood
28♀/55♂ 26♀/56♂
n: number of subjects and DNA samples; N: number of RNA samples; SD: Standard Deviation; ♀: female; ♂: male.
cording to the manufacturer's protocol. RNA was extracted from the blood samples using the RiboPure™ Blood Kit (Ambion, USA) according to the manufacturer's protocol. Total RNA was quantified using Spectrophotometer NanoDrop - 2000 (NANODROP, USA). Concentrations were adjusted and stored at -80ºC until use. In addition, 100ng of RNA was used for cDNA synthesis in High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems™, USA) following the protocol provided by the manufacturer. All cDNA was stored at -20ºC.
portions (%) for dichotomic variables. The statistical analysis to determine the differences between the group means and their associated procedures was performed using the ANOVA test. The ANOVA test observes the variance in a particular variable and partitions into components attributable to different sources of variation. One-way ANOVA evaluates whether the means of several groups are equal. Differences were considered significant with a p value less than 0.05. All analyses were performed using Statistical Package for the Social Sciences (SPSS), version 20.0.
Gene Expression Analysis
RESULTS
All gene expression was measured using qRT-PCR according to protocols established by Livak [27] in the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems™, USA). Cycling conditions were used as recommended by Applied Biosystems. We used APP (target; assay id: Hs01552282_m1), 18S (target; assay id: Hs03928990_g1*), B2M (endogenous control; assay id: Hs99999907_m1), UBC (endogenous control; assay id: Hs00824723_m1*) and GAPDH (endogenous control; assay id: Hs03929097_g1*) inventoried TaqMan® Gene Expression Assays and 28S (target; assay id: AIWR1RK) custom TaqMan® Gene Expression Assay. The amplification curve of each group was drawn, and the Ct values were successfully obtained for all genes (APP, 18S, 28S, B2M, UBC and GAPDH). Threshold values were uniformly set for all assays. All reactions were performed in duplicate. Replicates with standard deviations (SD) higher than 0.5 for the cycle threshold (CT) value were repeated or excluded from the analysis. All Ct values were obtained using the 7500 software 2.0, and these values were exported to the Excel software (Microsoft, USA) in order to calculate 2ΔCT and RQ. It is important to note that Relative Quantification (RQ) of APP, 18S and 28S genes were calculated using the healthy elderly group as a reference.
ApoE Genotyping In the brain samples, we compared the allele frequencies between elderly subjects (n=26) and AD patients (n=26). The E4 allele was found in 27% of patients with AD and 17.4% of subjects in the elderly control group; however, there were no significant differences in genotype distribution or/and allele frequency between the groups. Meanwhile, ApoE genotyping in blood samples and among the three groups (healthy young controls, healthy elderly controls and subjects with AD) revealed ApoE4 to be significantly associated with AD (p=0.0001). The allele frequencies and the genotype distribution are presented in Table 2 (Supplementary File). Amyloid Precursor Protein and Ribosomal Gene Expression in Peripheral Blood There was no difference between the groups in terms of the gene expression of the 18S ribosomal gene and amyloid precursor protein. However, the expression of 28S was significantly lower in patients with Alzheimer´s disease than in the other groups (p=0.0001) (Fig. 1). There was no difference in the expression of 28S between the young and elderly healthy subjects (p>0.05).
Statistical Analyses
Amyloid Precursor Protein and Ribosomal Gene Expression in Brain Tissue
Descriptive analyses were performed using means and standard deviations for continuous variables and with pro-
There was no difference in 18S and APP expression when different brain regions were compared (the entorhinal
4 Current Alzheimer Research, 2015, Vol. 12, No. 10
Table 2.
Tissue
Brain
Blood
Rasmussen et al.
Genotype and allele frequencies of ApoE polymorphism in subjects with Alzheimer Disease (AD) and Healthy elderly (HE) and Healthy young (HY) subjects. Groups
N
Genotypes %
Allele Frequencies %
E2/E2
E2/E3
E3/E3
E3/E4
E4/E4
E2/E4
E2
E3
E4
AD
26
-----
7.7
38.5
53.8
-----
-----
3.8
69.3
26,9
HE
26
-----
-----
61.5
23.2
-----
11.4
7.7
75.0
17.3
AD
82
-----
1.2
41.5
40.2
13.4
3.7
2.4
62.2
35.4*
HE
83
1.2
6.0
80.7
10.9
-----
1.2
4.9
89.1
6.0
HY
82
-----
24.4
52.4
20.8
1.2
1.2
12.8
75.0
12.2
N: number of individuals; *:Statistical difference when p < 0.05
cortex, the auditory cortex, and the hippocampus). However, when the total brain samples were analyzed collectively, gene expression differed significantly among the groups. APP mRNA was downregulated in AD patients, who were found to have an RQ of 0.93 compared to an RQ of 1.13 in the control group (p=0.021). Differences in APP gene expression were not observed among the groups when the entorhinal cortex (p=0.152), the auditory cortex (p=0.659), and the hippocampus were examined (p=0.165) (Fig. 2). There was a significant increase in 18S rRNA expression in AD patients (RQ=1.36) compared to the controls (RQ=1.09) (p=0.010) when all brain samples were analyzed collectively. However, this difference was not observed when each region was analyzed separately [entorhinal cortex (p=0.137), auditory cortex (p=0.327) and in hippocampus (p=0.109) (Fig. 3)].
(Fig. 4). There were no differences among the groups for 28S regulation in the auditory cortex (p=0.124).
Fig. (1). Comparison of the 2-ΔΔCT values of 28S rRNA gene, in blood samples, among in the Alzheimer’s disease patients group in relation to the healthy young and healthy elderly group. Bars represent means and error bars represent standard deviation. *p
