microRNAs expression correlates with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing

Journal Pre-proof microRNAs expression correlate with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing Juliana C.S. Chaves, Felippe T. Machado, Michael F. Almeida, Tatiana B. Bacovsky, Merari F.R. Ferrari

PII:

S0304-3940(19)30644-5

DOI:

https://doi.org/10.1016/j.neulet.2019.134541

Reference:

NSL 134541

To appear in:

Neuroscience Letters

Received Date:

16 December 2018

Revised Date:

28 August 2019

Accepted Date:

8 October 2019

Please cite this article as: Chaves JCS, Machado FT, Almeida MF, Bacovsky TB, Ferrari MFR, microRNAs expression correlate with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134541

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

microRNAs

expression

correlate

with

levels

of

APP,

DYRK1A,

hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing.

Juliana C.S. Chaves; Felippe T. Machado; Michael F. Almeida; Tatiana B. Bacovsky and Merari F. R. Ferrari*

Departamento de Genetica e Biologia Evolutiva, Instituto de Biociencias,

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* Corresponding Author:

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Universidade de Sao Paulo, Sao Paulo, SP, Brazil

Merari F. R. Ferrari, Ph.D.

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Associate professor

Universidade de São Paulo.

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Departamento de Genética e Biologia Evolutiva, Instituto de Biociencias,

Rua do Matao, 277, Cidade Universitaria.

Phone: +55 11 3091 8059 E-mail: [email protected]

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Sao Paulo, SP, 05508-090, Brasil.

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ORCID: http://orcid.org/0000-0001-7396-2196

Highlights

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APP expression increases as miRNAs decrease in older mice model of Down Syndrome.

•

miR-199b levels did not compensate increased DYRK1A mRNA in the hippocampus.

•

Hyperphosphorylated TAU212 levels correlate with increased Dyrk1A

levels.

Low expression of BDNF and high miR-26a and -26b were found in Down

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Syndrome.

ABSTRACT

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Down syndrome (DS) patients are more susceptible to Alzheimer’s disease (AD) due to the presence of three copies of genes on chromosome 21 such as

protein),

leading

to

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DYRK1A, which encodes a broad acting kinase, and APP (amyloid precursor formation

of

amyloid

beta

(Aβ)

peptide

and

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hyperphosphorylation of Tau. In this study, we investigated the association among miRNAs miR-17, -20a, -101, -106b, -199b, -26a, 26b and some of their target mRNAs such as APP, DYRK1A and BDNF, as well as the levels of hyperphosphorylated Tau in the hippocampus of a 2 and 5 months old mice model of trisomy 21 (Ts65Dn). Results indicated that increased APP expression in the

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hippocampus of 5 months old DS mice might be correlated with decrease in miR17, -20a, -101 and -106b. Whereas at 2 months of age normal levels of APP expression in the hippocampus was correlated with increased levels of miR-17, 101 and -106b in DS mice. DYRK1A mRNA also increased in the hippocampus of 5 months old DS mice and it is associated with decreased levels of miR-199b. Increased levels of DYRK1A in 5-month old mice are associated with increased phosphorylation of Tau at Thr212 residue but not at Ser199-202. Tau pathology is accompanied by decreased expression of BDNF and increased miR-26a/b in mice

of 5 months of age. Taken together, data indicate that miR-17, -20a, -26a/b, -101, 106b and -199b might be interesting targets to mitigate Tau and Aβ pathology in DS.

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Keywords: hyperphosporylation of Tau; Alzheimer’s disease; ageing.

Introduction

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Alzheimer's disease (AD), which is considered an age-related disease, is the most common dementia-related neurodegenerative disease in the world.

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Analysis of post-mortem brains from patients with AD revealed the presence of aggregates containing amyloid-beta (Aβ) peptide and neurofibrillary tangles formed

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by hyperphosphorylated Tau, both known as the main hallmarks of AD [17, 25, 30]. Tau is a microtubule associated protein (MAP) that stabilizes axonal microtubules mainly [28].

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Amyloid precursor protein (APP) is a highly conserved transmembrane protein. Cleavage of APP can occur by two pathways through α- or β- secretases which remove nearly the entire extracellular domain, while the membraneanchored C-terminal fragments are associated with intracellular signaling [31]. APP is subsequently cleaved by γ-secretase, a large multi-unit complex, within the

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plasma membrane. While cleavage of APP by α-secretase precludes the production of Aβ, cleavage by β- and γ-secretases results in the generation of Aβ and its subsequent extracellular deposit, which has a critical and early role in the pathogenesis of AD. About 50% of individuals with trisomy 21 (Down syndrome) develop AD after 50 years of age. Besides that, eventually all individuals with Down syndrome develop AD after they are 60 years old [9]. One possible association between

Down syndrome and AD may rely on DYRK1A gene [13] that is located on chromosome 21, and its triplication increases the expression and activity of DYRK1A, favoring phosphorylation of Tau in threonine 212 (Thr212) [21, 24]. High levels of hyperphosphorylated Tau at Thr212 contribute to intensify fibrillar tangles assembly in hippocampal neurons leading to cell death [1]. DYRK1A is a member of the dual-specificity tyrosine-phosphorylationregulated kinases family. Among DYRK1A functions it is the neuronal development and survival. In addition, DYRK1A gene plays a role in controlling the levels of APP

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and Tau proteins [20]. Furthermore, overexpression of DYRK1A gene enhances the levels of both proteins, contributing to neurodegeneration [27]. In cases of

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Down syndrome, DYRK1A expression is 50% increased causing phosphorylation of Tau protein and unbalance of the 4R proportion to 3R, which are the stable and

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unstable protein isoforms, respectively [10, 27]. The unbalance of Tau isoforms is associated with the formation of neurofibrillary tangles and thus protein

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aggregation [2]. On the other hand, decrease in levels of DYRK1A may prevent Tau aggregation [13].

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Protein expression levels may be regulated by microRNAs (miRNAs), which are a class of small (21 to 25 nucleotides) endogenous noncoding RNAs. They are associated with RNA-induced silencing complex (RISC), which drives the complex

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to messenger RNAs promoting their downregulation. Each miRNA has different RNA targets as they promote the regulation of many gene transcripts. Thus, changes in miRNA levels may exert influence on many genes of a system, which characterizes them as an important target in the study of cellular processes and eventually diseases such as AD [5, 18].

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In the central nervous system, miRNAs are involved with the establishment

and maintenance of cell identity, axonal guidance, neurogenesis control and synaptic plasticity [6]. Accordingly, the unbalance of miRNAs might contribute to the onset of neurodegenerative diseases.[14] Specifically in AD several miRNAs are involved in the expression of proteins associated with formation of Aβ deposits, hyperphosphorylation of Tau and growth factors such as the brain-derived neurotrophic factor (BDNF) [7].

BDNF is the major brain neurotrophin. It is involved in neuronal differentiation, maturation and survival; it also modulates synaptic transmission and neuronal plasticity [23]. Therefore, regulation of BDNF expression by microRNAs is crucial to neural development and survival. In mice, chromosome 16 is analogous to part of the human chromosome 21, and therefore, triplication of chromosome 16 provides a genetic model for Down Syndrome [3]. This mouse model also shares many characteristics of AD, such as

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accumulation of Tau in the hippocampus [12].

In view of this, the present study aimed at investigate the levels of miRNAs

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miR-17, -20a, -26a, -26b, -101, -106b and -199b, and some of their mRNA targets, such as APP, DYRK1A and BDNF, as well as the levels of hyperphosphorylated

Animals

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Materials and Methods

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Tau in the hippocampus of 2 and 5 months old mice model of trisomy 21.

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In this study we used 2- and 5-month old mice (Mus musculus) of B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ (The Jackson Laboratory, # 004850) and B6EiC3SnF1/J (The Jackson Laboratory, #001875) strains. Mice were kept in

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the animal facility of the Institute of Biosciences of the University of Sao Paulo, in light/dark cycle of 12 hours (lights on at 7am), with appropriate food pellets and water ad libitum.

The experiments presented herein were in accordance with the ethical aspects of animal experimentation recommended by Concea and according to the

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Federal Law No. 11,794. In addition, all experimental procedures were approved by the Ethics Committee on Animal Experimentation of the Institute of Biosciences (CEUA IBUSP Protocol #184/2013). Extraction of DNA and Genotyping All animals were genotyped to determine the group they belong to (Control or Down) according to the protocol provided by The Jackson Laboratory. Briefly, a fragment of approximately 5.0 mm of the tail end of each animal

was removed at the time of euthanasia. Total DNA was isolated by incubation with 500μl of lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, pH8.5) and 2.5μl proteinase K) at 55°C, during 40 minutes in a thermomixer at 1000 rpm of agitation. After that, the solution was vigorously homogenized and centrifuged (3000 rpm, 10 minutes) to separate the undigested material. The supernatant was transferred to a new tube to which was added 500 ul of isopropyl alcohol for DNA precipitation. This solution was centrifuged for 15 minutes at 12000 rpm. The precipitate was resuspended in 100 ul of TE buffer and quantified in a

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spectrophotometer (Epoch, Biotek).

PCR was carried out to identify the trisomy of chromosome 16. PCR

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solution contained 2.0 μl of DNA (5 ng/uL), 6.0 μl 2xPCR of master mix (Invitrogen), 0.1 μl of each control probe (20 μM, 1181 and 1182), 0.6 μl of each

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primer which recognizes the junction between chromosomes 16 and 17 (20 μM; oIMR7338 and oIMR7339), and water to complete 12 μl reaction.

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PCR products were visualized after electrophoresis in an agarose gel where 7.0 μl of PCR reaction were mixed with 2.0 μl of Blue Green reagent (LGC

specific of 275 bp).

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Biotechnology). Trisomic mice presented two bands (the control of 300 bp and the

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Dissection of hippocampus and extraction of RNA and protein Mice were deeply anesthetized with ketamine before decapitation, their brains were removed and dissected out to obtain the bilateral hippocampus. Total RNA was extracted (from one hippocampus side for 5-month old and from both sides of hippocampus for 2-month old mice) and enriched to small RNA

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using the mirVana kit (ThermoFisher Scientific). To access the quantity and quality of extracted RNA, samples were subjected to spectrophotometer to have the UV absorbance determined. Samples that revealed a ratio A260:A280 below than 1.8 were discarded. After determination of the RNA concentration, another sample containing 1 μg of total RNA and 1 μL of BlueGreen reagent (LGC) was fractionated on 1% agarose gel. Samples that presented the ratio 2:1 between 28S and 18S ribosomal RNA were used to real-time PCR analysis.

The other hippocampus side from 5-month old mice was subjected to protein extraction in a solution containing PBS, pH 7.4, with 1% NP40, 0.5% sodium deoxycholate, 1% SDS, 1 mM EDTA, 1 mM EGTA and 1% protease inhibitor cocktail (Sigma, St. Louis, MO). After centrifugation at 14000 rpm for 10 minutes, the resulting supernatant was fractionated by means of SDS-PAGE.

Reverse transcription and RT-PCR To 1 μg of total RNA, we added MgCl2 (5.5 mM), dNTPs (500 mM), random

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hexamers (2.5 mM), RNAse inhibitor (0.4 U/ml) and MultiScribe reverse transcriptase (1.25 U/ml) to a final volume of 50 μl. Primers, probes and reagents

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for real-time PCR were obtained from Applied Biosystems (TaqMan). The following RNAs were evaluated: APP mRNA (Mm01344172_m1), DYRK1A mRNA

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(Mm00432934_m1), BDNF mRNA (Mm04230607_s1), miR-17 (002543), miR-20a (002491), miR-106b (000442), miR-101 (02253), miR-199b (001131), miR-26a

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(463227_mat) and miR- 26b (000407). Protocol provided by the manufacturer was rigorously followed in which 12.5 μl of 2xPCR Master Mix, 1.25 μl of primers/probe

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and 6.25 μl of DEPC water were added to 5 μl of experimental cDNA. The expression of 18S rRNA was used as internal control to normalize the results across samples using the 2-ΔΔCT method and considering control 2-month old mice

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as reference, designated as 1 in graphs.

Western Blot

Protein samples (15μg) were applied to a 12% polyacrylamide gel for resolving at 100V during approximately 2 hours. Proteins were then transferred to

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nitrocellulose membrane for 1h at 100V. Blots were blocked and subjected to antibodies incubation. Membranes were blocked with milk 5% in TBT-T during 1 hour at room

temperature followed by incubation at room temperature, for 3 hours, with rabbitanti-DYRK1A (Santa Cruz Biotechnology, SC-2899, 1/1000), or for 1 hour with rabbit-anti-Tau 199-202 (Sigma-Aldrich, T5819, 1/1000), mouse-anti-Tau total (Invitrogen 44740, 1/1000) or mouse-anti-beta-actin (Santa Cruz Biotechnology,

sc-47778, 1/1000). Horseradish peroxidase-conjugated secondary antibody incubations were performed at room temperature for 1 hour with anti-rabbit 1/10000 (GE Healthcare, NA934, 1/10000) or anti-mouse (GE Healthcare, NA931,1/6000). Development was done after 5-minute incubation with enhanced chemiluminescence reagent (Millipore) and exposure to chemiluminescence sensitive films (Hyperfilm ECL, Amersham Biosciences). Films were quantified using Image J software (NIH). Normalization was done by dividing the values corresponding to the bands relative

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to proteins of interest by beta-actin values.

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Statistics

Results were analyzed by either two-way ANOVA followed by the Bonferroni

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post-test or Student’s T-test accessed through GraphPad Prism. A p-value ≤0.05 was considered to show statistically significant differences. Data are expressed as

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mean± standard error of the mean. N=5.

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RESULTS

APP mRNA expression increases as miR-17, -20a, -101 and -106b decrease in older mice model of Down Syndrome.

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There is no significant differences in the expression of APP mRNA between Down and control mice at the age of 2 months (Figure 1A). Five-month old Down mice present increased expression of APP mRNA in the hippocampus as compared with age-matched control mice (Figure 1A). All miRNAs significantly decreased in the hippocampus of 5-month old as compared with 2-month old Down

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mice reaching the levels of same-age control mice (Figure 1B-1E). Interestingly at 2 months of age, mice presented increased levels of miR-17, -101 and -106b in the hippocampus, which appears to be correlated with normal levels of APP mRNA at this age. Whereas, as the levels of these miRNAs drop, at the age of 5 months, APP mRNA rises (Figure 1).

miR-199b levels seem not compensate the increased DYRK1A mRNA

in the hippocampus during ageing. DYRK1A mRNA increased 3.5 fold in 5-month old DS mice compared with same-age control mice (Figure 2A), however the expression of its regulatory miRNA, miR199b, did not change compared with same-age control mice, indeed it decreased overtime in both control and Down mice (Figure 2B). Two-way ANOVA revealed that there is interaction between genetic background and age for DYRK1A expression, since only aged Down mice presented increase in DYRK1A mRNA levels. On the other hand, only age

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levels in Down mice because of the extra copy of DYRK1A.

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influences miR-199b expression, which contributes to the increased DYRK1A

levels in mice model of Down Syndrome.

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Hyperphosphorylated TAU212 levels correlate with increased DYRK1A

Five-month old mice did not present changes in expression of Total Tau

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protein (Figure 3A) as well as Tau hyperphosphorylated at serine 199 and 202 residues (Figure 3B). However Tau hyperphosphorylated at threonine 212 residue

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was significantly increased in the hippocampus of 5-month old mice as compared with control (Figure 3C). DYRK1A protein levels were 2-fold increased in the hippocampus of 5-month old DS mice (Figure 3D). These data correlates with

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DYRK1A specificity in phosphorylating Tau at threonine 212.

Decreased expression of BDNF mRNA is correlated with increase in miR-26a and -26b in hippocampus of a mice model of Down Syndrome. In our study, BDNF mRNA expression was decreased in the hippocampus

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of both control and Down mice at 5 months of age (Figure 4A). However, decrease in BDNF mRNA is more pronounced in Down (90% decrease) than in control mice (50% decrease). These data might correlate with the increased miR-26a/b expression in the mice model of Down syndrome (Figure 4B and 4C). Whilst control mice might have other mechanisms involved in BDNF repression.

DISCUSSION In this study it was demonstrated a possible correlation of APP, DYRK1A and BDNF mRNAs expression and some of their regulatory miRNAs in the hippocampus of 2- and 5-month old mice model of Down Syndrome. Data showed that, after 5 months of age, Down mice presented increased APP and DYRK1A mRNA levels, which was expected since these animals harbor 3 copies of these genes which are responsible for increased protein expression in adult DS brains

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compared to control groups [15, 19].

Results showed herein shed light on some cellular mechanisms underlying

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modulation of miRNAs upon key mRNAs related to Alzheimer’s disease. In fact many miRNAs are deregulated during neurodegeneration and are potential

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therapeutic targets for treating Alzheimer’s disease [11].

Moreover, ageing is an important risk factor for triggering neurodegenerative

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diseases. In the present study it was shown that young mice model of Down Syndrome present same levels of mRNAs and miRNAs evaluated herein, while

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most differences are observed in 5-month old Down mice. These evidences confirm that the B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ mouse is suitable for Alzheimer’s diseases studies.

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The present findings, together with previous studies that decribed the involvement of these miRNAs in preventing Aβ accumulation [16], allow us to hypothesize that increase in miR-17, -101 and 106b may counteract APP levels in young DS mice, while decrease in these miR at 5 months of age may contribute with the raise in APP mRNA level.

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Levels of miR-199b expression were decreased in both control and Down

mice after 5 months of age, indicating that this miR is regulated by age and may contribute to aggregation pathology during neurodegeneration, since DYRK1A is triplicated and miR-199b is not. DYRK1A is postulated as a kinase that acts upon hyperphosrylation of Tau, but is also localized to nucleus where it may interfere with Tau splicing, generating an imbalance between 3R- and 4R Tau [29], contributing to accumulation of Tau,

which is one of the hallmarks of Alzheimer’s disease. It has also been demonstrated that increased of Tau Thr 212 levels correlate with increased levels of DYRK1A [15]. Our findings suggest a physiological role of DYRK1A in Tau pathology, since an extra copy of DYRK1A gene might contribute to early onset of Alzheimer's disease and it is not counteracted by miR-199b. Analyses of total Tau and Tau Ser199-202 demonstrate that DYRK1A might not phosphorylate these residues. It was observed that BDNF mRNA levels decreased in DS mouse model of

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5 months of age, which may be related to memory deficits and decreased synaptic plasticity in these animals [4, 8]. Our results corroborated previous data indicating

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that decreased levels of BDNF is correlated with increase in miR-26a/b in the mice model of Down Syndrome [7].

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Although analysis of miRNAs regulation upon specific targets is difficult due to multiple miRNA targets, interrelation between miRNAs, clusters of miRNAs and

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a variety of miRNAs acting upon one single mRNA [22, 26], our data indicate that overexpression of DYRK1A contributes to Tau pathology in this mice model of

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Down Syndrome. In addition, our findings revealed that overexpression of this kinase might contribute to the early onset of neurodegeneration in Down syndrome. However, whether these changed miRNAs participate in the regulatory

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process of APP and DYRK1A expression in Down with 5-month-old and/or whether these changed miRNAs combination could be biomarkers to predict the early cognitive decline, still need further investigation as these miRNAs have different RNA targets and they may regulate the expression of many other genes.

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CONFLICT OF INTEREST

Authors declare no conflicts of interests regarding data presented in this

manuscript.

ACKNOWLEDGEMENTS

This study was supported by research grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (2015/18961-2; 2015/10892-1; 2018/07592-4), Conselho Nacional de desenvolvimento Cientifico e Tecnologico (CNPq) (471999/2013-0; 401670/2013-9) and Coordenação de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) (Finance code 001). J.C.S.C. and F.T.M. received fellowships from CNPq and FAPESP; M.F.A. and T.B.B. received

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fellowships from FAPESP.

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Figure Legends Figure 1. Relative expression of APP mRNA (A), miR-17 (B), miR-20a (C), miR101 (D) and miR-106b (E) in the hippocampus of 2- or 5-month old Down and control mice. Results are presented as mean ± SEM, fold change relative to 2month old control mice. *p <0.05; **p <0.01; ***p <0.001, compared with 2-month ##

p <0.01;

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p <0.001 compared

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old mice with same genetic background; # p <0.05;

with same-age control mice, according to two-way analysis of variance (two-way

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ANOVA) followed by Bonferroni post-test. N = 5.

Figure 2. Relative expression of DYRK1A mRNA (A) and its regulatory microRNA miR-199b (B) in the hippocampus of 2- or 5-month old Down and control mice. Results are presented as mean ± SEM, fold change relative to 2-month old control mice. **p <0.01 compared with 2-month old mice with same genetic background;

##

p

<0.01 compared with same-age control mice, according to two-way analysis of

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variance (two-way ANOVA) followed by Bonferroni post-test. N=5.

Figure 3. Levels of total Tau (A), hyperphosphorylated Tau at serine 199 and 202 residues (pTAU 199-202, B), hyperphosphorylated Tau at threonine 212 residue (pTAU 212, C) and DYRK1A (D) in the hippocampus of 5-month old mice of age compared to control normalized to beta-actin. Data are presented as percentage of

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control ± SEM, *p<0.05 compared with control, according to the Student's t test. N=5.

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Figure 4. Relative expression of BDNF mRNA (A) and its regulatory microRNAs miR-26a (B) and miR-26b (C) in the hippocampus of 2- or 5-month old Down and control mice. Results are presented as mean ± SEM, fold change relative to 2month old control mice. *p <0.05, **p <0.01 and ***p <0.001 compared with 2-month old mice with same genetic background; #p <0.05 compared with same-age control

mice according to two-way analysis of variance (two-way ANOVA) followed by

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Bonferroni post-test. N=5.