QFB Marisol Herrera-Rivero

UNIVERSITY OF VERACRUZ CENTRE OF BIOMEDICAL INVESTIGATIONS

Expression analysis of tau, APP, nicastrin, betasecretase and associated genes, in leukocytes, as markers of Alzheimer’s disease

THESIS

TO OBTAIN THE DEGREE OF:

Doctor in Biomedical Sciences

PRESENTS:

Q.F.B. Marisol Herrera-Rivero

Supervisor: Dr. Gonzalo Emiliano Aranda-Abreu

Xalapa, Ver., Mexico

November, 2013 CONACYT Scholarship #223277

Expression analysis of tau, APP, nicastrin, beta-secretase and associated genes, in leukocytes, as markers of Alzheimer’s disease - Summary of results

ACKNOWLEDGEMENTS

Very special thanks to all the people whose contributions made this work possible:

Gonzalo E. Aranda-Abreu, PhD

Centre of Cerebral Investigations, UV

Prof. Simon Lovestone

Institute of Psychiatry, King’s College London

Alison Baird, PhD


Stephen Newhouse, PhD


Michelle Lupton, PhD


Abdul Hye, PhD


Chantal Bazenet, PhD


M. Elena Hernández-Aguilar, PhD

Centre of Cerebral Investigations, UV

Abraham Soto-Cid, PhD

IMSS, Clinic 11

Rocío Coutiño-Rodríguez, PhD

Institute of Public Health, UV

Roberto Zenteno-Cuevas, PhD

Institute of Public Health, UV

Aleph A. Corona-Morales, PhD

General Direction of Investigations, UV

Enrique Juárez-Aguilar, PhD

Institute of Health Sciences, UV

Juan D. Santiago-García, PhD

Institute of Biological Investigations, UV

José Locia-Espinoza, MSc

Doctorate in Neuroaetiology, UV

Petty Lara-Ramos, BA

Residency “Emperatriz de las Américas, S.C”

Sister Bertha Criollo-Vázquez

Cáritas Xalapa

Rosa Elena Sánchez-Campos

Association “No Me Olvides Xalapa, A.C.”

And to all our donors and patients’ family members.

I also thank with great affection to mi classmates and friends: Erika Contreras, Montserrat Melgarejo, Guadalupe Cano and Sol de la Peña, as well as my friends: Francisco, Vichnu, Alejandro, Enrique, Celeste, Héctor, Liz Mariana, Leticia and Edgar, for all of their support.

I dedicate this work to the most important people in my life: my parents. Thank you for your love and support.

Marisol Herrera-Rivero

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ABSTRACT Tau, APP, nicastrin and β-secretase are the main molecules implicated in the pathophysiology of Alzheimer’s disease by being directly responsible for the generation of the amyloid plaques and neurofibrillary tangles. Several genetic variants and medical conditions common in the adult population have been linked to an increased risk to develop the disease. A peripheral immune response to changes in the Alzheimer’s brain also exists, although little is known about how tau, APP, nicastrin and β-secretase behave in non-neuronal cells. Easily accessible molecular markers for an early Alzheimer’s diagnosis would have important implications for disease treatment and for the rate of cognitive decline. We investigated the potential of tau, APP, nicastrin, β-secretase and related genes to diagnose Alzheimer’s disease using blood samples. In a small independent sample of cognitively healthy individuals, representing normal cognitive ageing, and patients with Alzheimer’s and other neuropathologies, we measured blood pressure and serum glucose, cholesterol and triglyceride levels as well as the gene expression of our four primary genes in lymphocytes through end-point RT-PCR. Also, we used a larger sample including control individuals and patients with Alzheimer’s and mild cognitive impairment to analyse microarray expression data of various genes in leukocytes and single polymorphisms influencing gene expression. Our results suggest there is no significant variation in gene expression through normal ageing; nevertheless, differences in gene expression and variability of tau, APP, nicastrin, β-secretase and related genes were identified between Alzheimer’s disease and controls as well as other neuropsychiatric pathologies, including mild cognitive impairment. We built Alzheimer’s disease classifiers using these results, identifying two models showing potential for the differential diagnosis of the disease using an innovative approach for variable selection. Overall, our results highlight the potential the expression of tau, APP, nicastrin, β-secretase and related genes have as molecular markers for the diagnosis of Alzheimer’s disease.


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JUSTIFICATION

Biomarker investigation for Alzheimer’s disease (AD) has strengthened in recent years due to the implications an early and accurate diagnosis may have on disease progression. A blood marker represents a cost-effective and accessible test, for which a number of molecules are being investigated in the blood of AD patients. Recently, studies in leukocytes have demonstrated that specific patterns for AD, compared with cognitively healthy individuals, can be identified. For this work, we aimed to analyse clinical variables associated with the risk to develop AD as well as tau, APP, nicastrin, βsecretase, pivotal genes in AD pathophysiology, and genes related to these by the interaction of their products or by the sharing of similar features, in order to investigate their potential use in the generation of tools aiming at the differential diagnosis of AD. To our knowledge, the approach of our study is different from studies by other research groups so far because, instead of focusing on those genes showing statistically significant variation in AD, compared to control groups, we investigated genes that are key to the neuropathology and genes relating to them, even when these may not share the same pathways. Therefore, we contribute an innovative approach for the generation of disease classifiers, not only for AD but for a diversity of disorders of similar complexity.

Hypothesis Expression of tau, APP, nicastrin, β-secretase and genes related to them, in peripheral leukocytes, presents specific patterns for Alzheimer’s disease with diagnostic use. General Objective Evaluate the potential of the expression of tau, APP, nicastrin, β-secretase and genes related to them, in leukocytes, as blood markers for the diagnosis of Alzheimer’s disease. Marisol Herrera-Rivero

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Specific Objectives

 Analyse the expression of tau, APP, nicastrin and β-secretase in lymphocytes throughout normal ageing, in Alzheimer’s disease and in non-Alzheimer’s neuropathologies.  Analyse the levels of blood pressure and serum glucose, total cholesterol and triglycerides throughout normal ageing, in Alzheimer’s disease and in nonAlzheimer’s neuropathologies, and identify their correlation with the expression of tau, APP, nicastrin and β-secretase in lymphocytes.  Analyse in leukocytes the expression of genes relating to tau, APP, nicastrin and βsecretase in patients with Alzheimer’s disease, mild cognitive impairment and cognitively healthy individuals.  Identify single nucleotide polymorphisms (SNPs) in tau, APP, nicastrin and βsecretase associated with Alzheimer’s disease and establish genetic differences between Alzheimer’s and mild cognitive impairment.  Identify expression quantitative trait loci (eQTLs) in leukocytes for tau, APP, nicastrin, β-secretase and the related genes, in patients with Alzheimer’s disease, mild cognitive impairment and cognitively healthy individuals.  Build and compare classifiers for Alzheimer’s disease on the basis of the results obtained from the different analyses performed.


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METHODS AND RESULTS

I. Expression of tau, APP, nicastrin and β-secretase in lymphocytes throughout normal ageing, in Alzheimer’s disease and in non-Alzheimer’s neuropathologies

Participants for a small independent sample were recruited in the Mexican cities of Xalapa, Veracruz, Cordoba and Puebla. Cognitively intact volunteers with an active lifestyle, no history of cerebrovascular events, traumatic brain injury or neurological disease and showing no signs of cognitive impairment or memory problems were classified in four groups by age range as a model of normal ageing (Table 1). An AD group and a group of mixed non-AD neuropsychiatric disorders (NP) were formed including patients previously diagnosed in the private practice by appropriate health professionals.

Table 1. Classification of individuals in the independent sample. Group I II III IV AD NP

Condition Cognitively healthy Cognitively healthy Cognitively healthy Cognitively healthy Possible/Probable AD Non-AD neuropsychiatric pathology

Age Range 20 – 35 36 – 50 51 – 65 > 65 > 65 > 65

In total, 86 individuals were initially included in the independent sample. For all of them, blood pressure was measured and blood samples were collected after an informed consent form was signed by either the volunteer or a patient’s family member. Participants were instructed to present with 12h fasting and in absence of any known inflammatory/immune conditions. Glucose, total cholesterol and triglyceride levels were quantified in serum samples using colorimetric procedures and all four clinical variables were classified as “normal” or “elevated” according to the standard reference values Marisol Herrera-Rivero

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used in the clinical laboratory in Mexico. Lymphocytes were isolated from whole peripheral blood to extract total RNA and carry out end-point RT-PCR reactions to amplify MAPT, APP, NCSTN, BACE1 and GAPDH as the housekeeping gene (Figure 1A). Tau was not detected in the conditions of the present study, but for two samples initially corresponding to group II. Therefore we tested the PCR primers in LNCaP cells to corroborate primers and reaction conditions were working appropriately (Figure 1B). Phosphorylated tau proteins were not detected by means of western blotting in any sample. A further interview with the participants in whose samples we detected a very mild PCR signal for tau revealed they appeared to have been showing signs of inflammatory processes unidentified at the time of sample collection. Nevertheless, we found this observation interesting for future studies as we believe acute but not the silent, chronic inflammatory processes involved in neuropathologies may induce tau expression in peripheral lymphocytes.

A

B

Figure 1. A) PCR products for MAPT, APP, NCSTN, BACE1 and GAPDH in lymphocytes. B) PCR product for MAPT in LNCaP cells.

For individuals with a questionable diagnosis or showing signs of ongoing inflammatory/immune processes within 48h after sample collection, as well as for samples of poor quantity and/or quality, data was disregarded prior to statistical analysis. 72 samples were considered suitable for the final analyses (Table 2). Marisol Herrera-Rivero

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Table 2. Details of individuals in the independent sample. I

II

III

IV

EA

PN

12

12

12

12

12

12

30 ± 1

44 ± 1

58 ± 1

80 ± 2

80 ± 2

78 ± 2

Males

4

4

7

6

2

5

Females

8

8

5

6

10

7

Sample number Mean age

Neuropathologies in NP:

Lesion by cerebrovascular event

4

Parkinson’s disease

4

Psychiatric disorders

2

Mild cognitive impairment

1

Vascular dementia

1

Statistical differences were not found for the expression of APP, NCSTN or BACE1 between genres or for the interaction between group and genre in our model of normal ageing, suggesting the expression of these genes in peripheral lymphocytes remains statistically very similar regardless of sex and age. For APP and BACE1, expression levels in lymphocytes appear to be conserved as well despite of cognitive status. However, we observed APP tends to be expressed at higher levels than BACE1, except in the AD group where APP and BACE1 expression levels are much more similar than in all other groups (Figure 2). We believe this may suggest an imbalance occurs in AD which, although not statistically significant, may contribute useful information for the differential diagnosis when combined with other variables. NCSTN was not detected in the AD group and detected in all NP samples; nevertheless, for the case of the normal ageing we detected NCSTN in some samples and did not in some others, in all four groups, which we hypothesized could be caused by genetic polymorphisms common in our population and located within the sequence fragment where one of the selected primers aligns. This hypothesis considers that other groups have reported an increased NCSTN expression in AD leukocytes in different populations, detected by different methodologies, and that our primers and reaction conditions were working properly. Thus we believe NCSTN was expressed in our Marisol Herrera-Rivero

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samples but an unknown factor interfered with our ability to visualize this expression. A missense single mutation in the region of alignment with our primer reverse was reported in the SNPdb in 2012, which made us consider to further study our observation by sequencing experiments to investigate whether this or a similar genetic variation prevented us from detecting NCSTN in lymphocytes of AD and other control individuals. To be the case, this variation may be associated with AD in our Latin American population and thus we consider this to be a very important result from our study, although we are aware of the limitations of the study and that further work remains to be done regarding this observation. As expected, NCSTN was the only expression variable showing statistical significance when compared against group IV as a normal control and group NP as non-AD neuropathology control (Figure 2).

Relative gene expression

0.5 0.4

APP NCT BACE1

0.3 0.2 0.1 0 I

II

III Group

IV

AD

NP

Figure 2. Relative gene expression of APP, NCSTN and BACE1 in peripheral lymphocytes from the independent sample. Only NCSTN showed statistical difference (p