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Rapamycin rejuvenates oral health in aging mice

Citation

Kerns, Kristopher (2020), Rapamycin rejuvenates oral health in aging mice, Dryad, Dataset, https://doi.org/10.5061/dryad.f4qrfj6sn

Abstract

Periodontal disease is an age-associated disorder clinically defined by periodontal bone loss, inflammation of the specialized tissues that surround and support the tooth, and microbiome dysbiosis. Currently, there is no therapy for reversing periodontal disease, and treatment is generally restricted to preventive measures or tooth extraction. The FDA-approved drug rapamycin slows aging and extends lifespan in multiple organisms, including mice. Here we demonstrate that short-term treatment with rapamycin rejuvenates the aged oral cavity of elderly mice, including regeneration of periodontal bone, attenuation of gingival and periodontal bone inflammation, and revertive shift of the oral microbiome toward a more youthful composition. This provides a geroscience strategy to potentially rejuvenate oral health and reverse periodontal disease in the elderly.

Methods

DNA extraction

Mandible samples were cryogrinded and homogenized using Evolution with bead-beating tubes and ceramic beads (Bertin Instruments, Rockville, MD). Bacterial genomic DNA was extracted using the QIAamp DNA Microbiome Kit (Qiagen, Hilden, Germany) and further purified and concentrated using DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol, then stored at - 80°C until all samples were collected

Sequencing

The V3-V4 variable region of the 16s ribosomal RNA gene was amplified using gene-specific primers with Illumina adapter overhang sequences (5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ and 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’). Each reaction mixture contained 2.5 µl of genomic DNA, 5 µl of each 1 µM primer, and 12.5 µl of KAPA HiFi HotStart ReadyMix. Amplicon PCR was carried out as follows: denaturation at 95°C for 3 minutes, 35-40 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, followed by a final extension step at 72°C for 5 min. PCR products were verified using gel electrophoresis (1% agarose gel) and cleaned with AMPure XP beads (Agencourt, Beckman Coulter Inc., Pasadena, CA, USA). Amplicons were then indexed using the Nextera XT Index Kit V2 set A and set D (Illumina) and purified again with AMPure XP beads to remove low molecular weight primers and primer-dimer sequences. DNA concentrations were concentration of 1-2nM using the SequalPrep Normalization Kit (Invitrogen). Samples were pooled into a single library which was analyzed using the TapeStation 4200 High Sensitivity D1000 assay (Agilent Technologies, Waldbronn, Germany) and Qubit High Sensitivity dsDNA assay (Thermo Fischer Scientific) to assess DNA quality and quantity. The final pooled library was then loaded on to an Illumina MiSeq sequencer with 10% PhiX spike, which served as an internal control to balance for possible low diversity and base bias present in the 16S amplicon samples, and was run for 478 cycles and generated a total of 5.68 million paired-end reads (2×239 bp).

Bioinformatics

Raw paired-end sequences were imported in to Qiime2 (v. 2019.1) and were trimmed by 15 nt from the 5’ end and truncated to 239 nt for the 3’ end for both the forward and reverse reads respectively. The trimmed reads were then demultiplexed and denoised using the DADA2 package. Forward reads were only used in our analysis. Taxonomy was then assigned using the feature-classifier suite trained on the and the Human Oral Microbiome Database (HOMD v. 15.1). Samples were then filtered for taxonomic contaminants excluded samples with less than 10,000 reads. Alpha and Beta diversity as well as other analysis were done in R-Studio using the Phyloseq, Clustvis, ggplot2,  ampvis2, vegan, ade4 packages as part of the R suite.

Supplemental methods

Taxonomy filtered from samples was determined by analysis of kit controls with no template and zymo sequencing controls of know diversity and abundance (the QIAamp DNA Microbiome Kit (Qiagen, Hilden, Germany) DNA Clean and Concentrator Kit (Zymo Research). The following taxonomic assignments were removed as part of the dada2 workflow: Unassigned, Cyanobacteria, acidovorans, pestis, coli, flavescens, sakazakii, durans, diminuta, anthropi, monocytogenes, parasanquinis_clade_411, otitidis, subtilis, aeruginosa, fermentum.

Statistical Analysis

Results for the 16s rRNA sequencing, to identify statistically significant differences among agglomerated and normalized amplicon sequence variants (ASV) between samples as well as differences in alpha and beta diversity measures, we applied both the unpaired Wilcoxon rank-sum test as well as the two-tailed paired t-test – both with a 95% confidence interval (α = 0.05). Alpha diversity was assessed measuring Shannon, Chao1, Observed (ASV), and Fisher diversity measures. Beta diversity was measured using weighted Unifrac distances. Statistical analysis for the microbiome analysis was completed in R (v. 3.5.3).

Usage Notes

Workflow materials and R Markdown are provided in detail on the McLean Lab Github repository: https://github.com/kkerns85/Rapamycin_rejuvenates_oral_health_in_aging_mice

A web version of the R Markdown is available at: https://rpubs.com/kkerns85/Rapamycin_Rmrkdown

Raw read data is available on the European Nucleotide Archive (ENA) under study accession no. PRJEB35672

Funding

National Institute of Dental and Craniofacial Research, Award: DE027254

National Institute of Dental and Craniofacial Research, Award: DE023810

National Institute of Dental and Craniofacial Research, Award: DE020102

National Institute on Aging, Award: R01AG054180

National Center for Advancing Translational Sciences, Award: TL1 TR002318