Assessment of GAPDH expression by quantitative real time PCR in blood of Moroccan AD cases

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Review article

Assessment of GAPDH expression by quantitative real time PCR in blood of Moroccan AD cases

Nadia El Kadmiri

^a,b,^⇑

, Meryam El Khachibi

^b

, Ilham Slassi

^b,c

, Bouchra El Moutawakil

^b,c

, Sellama Nadifi

^b,1

, Abdelaziz Soukri

^d,1

aIBN ZOHR University, Faculté Polydisciplinaire de Taroudant, B.P: 271, 83 000 Taroudant, Morocco

bHassan II University of Casablanca, Laboratory of Medical Genetics and Molecular Pathology, Faculty of Medicine and Pharmacy, 19 Rue Tarik Ibnou Ziad, B.P: 9154, Morocco

cNeurology Department, IBN ROCHD University, Hospital, rue des Hôpitaux, Casablanca, Morocco

dLaboratory of Physiology and Molecular Genetics, Faculty of Sciences Aïn Chock, Hassan II University, Casablanca, Morocco

a r t i c l e i n f o

Article history:

Received 10 October 2016 Accepted 27 December 2016 Available online xxxx

Keywords:

GAPDH

Moroccan AD cases mRNA expression

a b s t r a c t

Introduction: Neuroproteomics studies have showed the high affinity interactions between GAPDH –b- amyloid in Alzheimer disease. The aim of our study is to complete our previous studies by assessing the mechanism responsible of decreased expression of GAPDH protein in the blood of Moroccan AD cases probably due to an alteration at the transcriptional level or at the post translational level.Methods: The mRNA expression of GAPDH was assessed by quantitative real time PCR in AD cases and healthy controls.

Results: Our result revealed a significant difference of mRNA expression level of GAPDH in AD cases as compared to healthy controls (P< 0.05).Conclusion: This data is consistent with several studies by showing the direct involvement of GAPDH in amyloid aggregation by undergoing several modifications, which influence its chemical structure and its biological activity.

1. Introduction

Alzheimer disease (AD) is the most common form of neurodegenerative illness[1]. This form of dementia leads to a progressive loss of memory and gradual decline in cognitive functions. Neu- ropathologically, two kinds of neuropathologic changes have been identified as major hallmarks in brain of AD patients: senile plaques deposition and neurofibrillary tangles accumulation. How- ever, a definitive diagnosis remains based on postmortem investigation[2–4]. To date, several mutations in amyloid precursor protein (APP) and the presenilins causing early-onset familial AD (EOAD) have been identified. (available at: http://molgen www.uia.ac.be/ADmutations). Proteomics studies have been conducted to identify proteins affecting the degree of neurodegeneration and could contribute to discover and define predictive biomarker signatures for AD[5–8].

Neuroproteomics studies performed recently, have assessed the potential involvement of the oxidoreductase, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), in the mechanism of neurodegeneration by showing the direct interaction between GAPDH and

neurodegenerative disease-associated proteins such as beta- amyloid (Ab) precursor protein (AbPP), Aband neurofibrillary tangles, with a high affinity, specifically in brain specimens. In AD, GAPDH can undergo several modifications in brains of AD patients, which fundamentally affect its chemical structure and its biological function[9–12].

In our previous findings, we elucidated the critical role of GAPDH and its interaction withb-amyloid in the blood of Moroc- can FAD cases carrying presenilin mutations. The activity of GAPDH in crude extracts from both the peripheral blood and brain specimens from Moroccan FAD cases carrying presenilin mutations was significantly decreased as compared to healthy controls. The protein expression level of GAPDH in brain specimens from mutant tau transgenic mice and patients with FAD was unchanged as compared to healthy controls. In contrast, the protein expression level of GAPDH in blood samples from patients with FAD was decreased as compared to healthy controls. Moreover there is an accumulation ofb-amyloid aggregates in the blood samples of patients with FAD and an increase in amyloid fibrils in both the blood and brain samples of these patients[13,14].

However, it remains to assess the mRNA expression of GAPDH in the blood of AD cases. Indeed this study was conducted, to eval- uate the mechanism responsible for the decreased expression of the protein, probably due to an alteration at the transcriptional level or at the post translational level.

⇑ Corresponding author at: Université Ibn Zohr, Faculté Polydisciplinaire de Taroudant, Hay El Mohammadi (Lastah) B.P: 271, 83 000 Taroudant, Morocco.

E-mail address:elkadmiri1979@gmail.com(N. El Kadmiri).

1 Equal contributors.

Journal of Clinical Neuroscience xxx (2017) xxx–xxx

Contents lists available atScienceDirect

Journal of Clinical Neuroscience

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j o c n

Please cite this article in press as: El Kadmiri N et al. Assessment of GAPDH expression by quantitative real time PCR in blood of Moroccan AD cases. J Clin Neurosci (2017),http://dx.doi.org/10.1016/j.jocn.2016.12.007

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2. Methods

2.1. Patients recruitment

Peripheral blood was taken from 15 AD cases and of 18 healthy controls. All patients were seen by the memory consultation group at the CHU IBN ROCHD Neurology Department in Casablanca, Mor- occo. All patients underwent standard somatic neurological exam- ination, cognitive function assessment, brain imaging, and laboratory tests. The protocol was approved by the Human Ethical Committee of the CHU IBN ROCHD in accordance with the Declara- tion of Helsinki for experiments involving humans. A written consent was obtained from the patients and their legal guardians prior to their enrollment in the study.

2.2. Real-time PCR analysis

Total RNA was extracted from peripheral blood. We performed the extraction by using trizol (Invitrogen) according to manufacturer’s instructions. The amount of total RNA was determined by measuring the absorbance at 260 nm (A260), using the NanoVue^TM Plus Spectrophotometer (GE Healthcare, UK). RNA samples were stored at 80°C until conversion into cDNA.

Reverse transcription was performed using 2

l

g of RNA, 1

l

^{l of}

random Examer (Invitrogen), 1

l

l of RT Superscript at 200 U/l (Invitrogen), 10

l

M dNTP (Invitrogen), and 4% of MMLV reverse transcriptase (Invitrogen).

In a final volume of 25

l

^{l, 2}

l

l per well of cDNA (50 ng) was used along with: 5reaction buffer, 10

l

M primers 1.5 mM MgCl2, 0.25 U Taq polymerase (Qiagen, London, UK). The thermal cycling was programmed: 35 cycles of DNA denaturation (1 min at 94°C), primer hybridization (1 min at 60°C), and elongation (1 min at 72°C) followed by a final elongation step (10 min at 72°C). Separation by electrophoresis on 2% agarose gel was performed and visualized using Ethidium Bromide straining. Samples for each patient and control were analyzed in duplicates to make sure products from all PCR runs were cross comparable. Real time PCR Applied Biosystem FAST 7500 apparatus and SYBR Green were used according to the manufacturer’s procedure. To determine fold changes, we use theDD^{CT method}[15]. Beta actin was used as a housekeeping gene.

2.3. Statistical analysis

The data were analyzed using the Studentt-test to compare the groups (control vs cases), with the level of significance set at P< 0.05. Statistical analyses were performed using the Graph PRISM V 6.01 software package.

3. Results and discussion

As shown in theFig. 1, Statistical analyses revealed a significant difference of mRNA expression level of GAPDH between AD cases and healthy controls (P< 0.05;P= 0.002). Our finding of quantitative real time PCR analysis revealed that the mRNA expression was altered indicating that the GAPDH was probably altered at transcriptional level. Several neuroproteomics studies have con- firmed that GAPDH in AD can undergo several different oxidative modifications, which modify its chemical structure and affect its biological activity. In brains of AD individuals, GAPDH binds to amyloid plaques and even NFTs. It was identified as a major component in this complex aggregates. The GAPDH forms an intermolecular disulfide bonds to interact with beta amyloid[16–18].

The interaction between GAPDH and beta amyloid leads to a dena- tured form of GAPDH. Therefore, the process of oxidation and

denaturation confer to GAPDH the ability to form complexes highly stable with Ab, which highlight the direct involvement of GAPDH in amyloid aggregation[19–22]. Cumming and Schubert[23], conducted a study on focusing on oxidative stress induced by Abin aggregation mechanism. This process promotes an increase in GAPDH intermolecular disulfide bonding in extracts of brain from AD cases. Therefore, it’s directly stimulates nuclear translocation and pro-apoptotic action of GAPDH, which increase cytotoxicity, decrease its enzymatic activity and indirectly raises apoptosis. Fur- thermore, Schulze et al.[24], demonstrated by gel filtration, the direct interaction between GAPDH and AbPP. Studies performed in this area mentioned that brain-derived GAPDH binds to a variety of Abisoforms, with high affinity for Ab(1–42). As known, GAPDH is also involved apoptosis cascades initiation[20–22,25].

Our result showed a decreased mRNA expression of GAPDH in AD cases assessed by qRT-PCR, which correlate to our previous finding that revealed a significant decrease in GAPDH protein expression in blood samples from AD cases assessed by Western blot[13].

Our finding provide the potential role of GAPDH in neurodegeneration by undergoing probably a transcriptional modification. In AD cases, the mRNA expression of GAPDH assessed by qRT PCR was affected, which leads to a decrease protein expression and a decrease enzymatic activity. According to other studies, our result reveals the interaction between GAPDH and Ab. This interaction modulates its expression and inhibiting its activity.

4. Conclusion

This data highlights the potential involvement of the GAPDH, in neurodegeneration. Therefore, we suggest that GAPDH interacts directly with Ab. All findings open prospects to clarify more these mechanisms in blood of AD cases by aiming to use GAPDH as a biomarker for predicting and monitoring AD modification.

Ethics statement

The protocol was approved by the Human Ethical Committee of the CHU IBN ROCHD inaccordance with the Declaration of Helsinki for experiments involving humans.

Consent

A written consent was obtained from the patients and their legal guardians prior to their enrollment in the study.

Fig. 1.Quantitative real time PCR analysis of mRNA expression level of GAPDH in peripheral blood from 15 AD cases and from 18 healthy controls. Data is expressed as 2^-DCT. A Significant difference of mRNA expression level of GAPDH (P< 0.05).

2 N. El Kadmiri et al. / Journal of Clinical Neuroscience xxx (2017) xxx–xxx

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Support sources

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

No additional external funding was received for this study.

Conflict of interest

The authors declare that no competing interests exist.

Acknowledgments

We would like to thank the staff of Genetics and Molecular Pathology Laboratory at the Faculty of Medicine and Pharmacy, Casablanca, Morocco.

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