Pantethine, an anti-cholesterol drug, was shown to inhibit the Ab (amyloid beta) peptide-induced expression of IL-1b in primary culture astrocytes derived from either untreated Ab-treated and untreated 5XFAD or Ab-treated WT. These observations prompted us to investigate the effects of pantethine at the transcriptomic level in the mouse AD model.

Transcriptomic effects of long-term pantethine treatment in the mouse Alzheimer's disease 5XFAD model

Illumina MouseRef-8 v1.1 Expression BeadChip

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disease

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Pantethine, an anti-cholesterol drug, was shown to inhibit the Ab (amyloid beta) peptide-induced expression of IL-1b in primary culture astrocytes derived from either untreated Ab-treated and untreated 5XFAD or Ab-treated WT. These observations prompted us to investigate the effects of pantethine at the transcriptomic level in the mouse AD model.


We used 5XFAD transgenic mice, which overexpress two human transgenes bearing five mutations linked to familial AD: APP (Swedish mutation K670N, M671L; Florida mutation I716V; London mutation V717I) and presenilin 1 (PSEN1; M146L, L286V), under transcriptional control of the mouse Thy1 promoter. These mice exhibit AD-related symptoms earlier than other animal models and amyloid deposition start in the cortex and subiculum at two months of age.Heterozygous male 5XFAD transgenic animals (Tg) and WT controls were obtained after breeding in our animal house facility. Animals were housed in cages in a controlled environment (22-25°C, 50% humidity and a 12-hour light/dark cycle) with free access to standard laboratory diet and water. At 1.5 month, Tg and WT mice were each separated into treated and vehicle groups (n=10 per group).At the age of 7 months, the animals were submitted to the intrudal aggressivity test and then sacrificed. A pool of 3 hippocampi for each condition (n=3) : Tg (vehicule treated), Tg+P (pantethine treated), WT (vehicule treated) and WT +P (pantethine treated) were used for RNA extraction. A total of 12 biological samples were therefore processed for the microarray experiment.

3 hippocampi from each experimental group (n=3 per group) were pooled and quickly snap-frozen in liquid nitrogen and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instruction. RNA concentration was determined using nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Villebon sur Yvette, France) and RNA integrity assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Les Ulis, France).

The mouse WG-6 expression BeadChip (Illumina Inc., Cambridge, UK), comprising 47,232 transcripts was used to generate gene expression profiles and hybridized to cRNAs, which were synthesized, amplified and purified using the Illumina TotalPrep RNA Amplification Kit (Ambion, Foster City, CA, USA) following manufacturer recommendations. Briefly, 200 ng of RNA were reverse transcribed. After second strand synthesis, the cDNA was transcribed in vitro and cRNA labelled with biotin-16-UTP.

Labelled probe hybridization to the Beadchips was carried out using Illumina’s protocol.

The Beadchips were scanned on the Illumina IScan using Illumina IScan image data acquisition software.

Based on a previous study, Tg and WT treated mice were given each an intraperitoneal injection of 15 mg of pantethine diluted in saline, two times a week for 5.5 months; vehicle animals received saline injections as control. Pantethine was purchased from Sigma-Aldrich (Saint Quentin-Fallavier, France). Purity of the preparation was ascertained using high performance liquid chromatography-coupled mass spectrometry (HPLC-LC-UV-MSD) and the purity of the compound reached 99%.

The Beadchips were scanned on the Illumina IScan using Illumina IScan image data acquisition software. Illumina GenomeStudio software and LumiR (www.bioconductor.org) were used for preliminary data analysis, data normalization and quality controls.

Mice were anesthetized with pentobarbital (0.36 g per kg) and transcardially perfused with 50 ml of NaCl 0.9%. Brains were microdissected and hippocampi collected for the assays. All experimental procedures were approved by the Ethics Committee of the Medical Faculty of Marseille and were carried out in accordance with the guidelines published in the European Communities Council Directive of November 24, 1986 (86/609/EEC). All efforts were made to reduce animal suffering and the number of mice.

Transcriptomic effects of long-term pantethine treatment in the mouse Alzheimer's disease 5XFAD model

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