Rapamycin rejuvenates oral health in aging mice

  1. Jonathan Y An
  2. Kristopher A Kerns
  3. Andrew Ouellette
  4. Laura Robinson
  5. H Douglas Morris
  6. Catherine Kaczorowski
  7. So-Il Park
  8. Title Mekvanich
  9. Alex Kang
  10. Jeffrey S McLean
  11. Timothy C Cox
  12. Matt Kaeberlein  Is a corresponding author
  1. Department of Oral Health Sciences, University of Washington, United States
  2. Department of Pathology, University of Washington, United States
  3. Center of Excellence in Maternal and Child Health, University of Washington, United States
  4. The Jackson Laboratory, United States
  5. Department of Periodontics, University of Washington, United States
  6. Department of Pediatrics, University of Washington, Seattle Children’s Research Institute, United States
5 figures, 1 table and 1 additional file

Figures

Cross-institution experimental design and assay for measuring periodontal bone loss.

(A) The NIA-UW colonies were received directly from the NIA Aged Rodent Colony at 4, 11, and 18 months, then acclimated for two months within the UW facilities (ARCF) until they reached 6 (Young), …

Rapamycin reverses age-associated periodontal bone loss (NIA-UW and JAX).

(A and B) Representative images of NIA-UW (A) maxillary and (B) mandibular teeth of Young, Old, and Old treated with 42ppm eRAPA (rapamycin) revealing age-associated periodontal bone loss. 8 weeks …

Rapamycin attenuates age-associated increase in RANKL expression and TRAP+ cells in periodontal bone.

(A and B) RANKL and OPG expression was determined by western blot analysis of total lysates from the periodontal bone of aged animals (Young, Adult, and Old) and Old animals treated for 8 weeks with …

Rapamycin alters increased NF-κB expression and inflammatory cytokine profiles in periodontium.

NF-κB p65 and IκBα expression was determined by western blot analysis of total lysates from the gingiva (A,C) and periodontal bone (B,D) of control animals (Young, Adult, and Old) and Old animals …

Figure 5 with 4 supplements
Rapamycin shifts aged oral microbiome towards young oral microbiome.

(A) Alpha diversity for all samples reveal significant differences between young (Y) and old (O) mice without rapamycin treatment (p<0.05). (B) Phylum level abundance using normalized agglomerated …

Figure 5—figure supplement 1
Independent Alpha Diversity Analysis for JAX and UW-NIA animals.

(A) Alpha Diversity measures for JAX Lab animals, including 6 month old (Y), 22 month old (O), and 22-month-old mice treated with rapamycin (R). (B) Alpha Diversity Measures for UW-NIA animals, …

Figure 5—figure supplement 2
Independent phylum level abundance analysis for JAX and UW-NIA animals.

Normalized agglomerated data were used to evaluate phylum level abundances using an amplicon sequence variant (ASV) approach for both JAX and UW-NIA animals independently. (A) Significant …

Figure 5—figure supplement 3
Independent beta diversity for JAX and UW-NIA animals by principal coordinate analysis using weighted unifrac distances.

(A) Beta diversity for only JAX samples there was significant separation between O and R (p<0.01; Axis 1, 69.8%) as well as between Y and O groups (p<0.05; Axis 2, 9.0%). (B) Beta diversity for only …

Figure 5—figure supplement 4
Combined beta diversity for JAX and NIA-UW animals by principal coordinate analysis using weighted unifrac distances by site location and group designation.

No distinct differences in clustering by site location was observed. A PERMANOVA (nperm = 999) analysis was performed using the Adonis package (v 2.5–6) as part of the R suite comparing site …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background(M. musculus)C57BL/6NIANIA Aged Rodent ColonyPMID:27549339
Strain, strain background(M. musculus)C57BL/6JJackson LaboratoryRRID:IMSR_JAX:000664
Chemical compound, drugRapamycinRapamycin HoldingsAmount based upon active rapamycin content to provide 42 parts per million concentration in chow.
Chemical compound, drugEudragitRapamycin Holdings
Chemical compound, drugRIPA Lysis and Extraction BufferThermoFisher
Scientific
Cat#: 89901
Chemical compound, drugHALT Protease
Inhibitor Cocktail
ThermoFisher
Scientific
Cat#: 78438
Chemical compound, drugHALT Phosphatase Inhibitor CocktailThermoFisher
Scientific
Cat#: 78420
Chemical compound, drugRestore Plus Stripping BufferThermfisher ScientificCat#: 46430
Antibodyanti -NFkBp65(Rabbit Monoclonal)Cell SignalingCat#: 8242WB (1:1000)
Antibodyanti-phospho-IκBα (Mouse monoclonal)Santa Cruz
Biotechnology
Cat#: sc8404WB (1:1000)
AntibodyAnti- IκBα
(Rabbit Monoclonal)
AbcamCat#: 32518WB (1:1000)
Antibodyanti-GAPDH
(Rabbit monoclonal)
Cell SignalingCat#: 5174WB (1:1000)
Antibodyanti-RANKL
(Mouse monoclonal)
Santa Cruz BiotechnologyCat#: sc377079WB (1:1000)
Antibodyanti-OPG
(Goat polyclonal)
R and D SystemsAF459WB (1:1000)
Antibodyanti-IgGκ
(Mouse monoclonal)
Santa Cruz BiotechnologyCat#: sc516102WB (1:10000)
Antibodyanti-rabbit IgG (Donkey polyclonal)Thermo Fisher ScientificCat#: 31458WB (1:10000)
Antibodyanti-goat IgG
(Donkey polyclonal)
AbcamCat#: ab97110WB (1:10000)
Commercial assay, kitProteome Profiler Mouse XL Cytokine ArrayR and D SystemsCat#: ARY028
Commercial assay, kitAcid Phosphatase, Leukocyte (TRAP) KitMillipore SigmaCat#: 387A-1KT
Commercial assay, kitQIAamp DNA Microbiome KitQiagenCat#: 51704
Commercial assay, kitDNA Clean and Concentrator KitZymo ResearchCat#: D4014
Commercial assay, kitKAPA HiFi HotStart ReadyMixKAPA BiosystemsCat#: KK2601
Commercial assay, kitNextera XT Index Kit V2IlluminaSet A: FC-131–2001
Set D: FC-131–2004
Commercial assay, kitAMPure XP BeadsAgencourtA63881
Commercial assay, kitSequalPrep Normalization KitInvitrogenA1051001
Commercial assay, kitTapeStation 4200 High Sensitivity D1000 assayAgilent TechnologiesG2991AA
Commercial assay, kitTapestation
Reagents
Agilent Technologies5067–5585
Commercial assay, kitHigh Sensitivity D1000 ScreenTapeAgilent Technologies5067–5584
Commercial assay, kitQubit High Sensitivity dsDNA assayThermoFisher ScientificQ32854
Commercial assay, kitMiSeq Reagent Kit v3 (600 cycle)IlluminaCat#: MS-102–3003
Commercial assay, kitPhiX Control Kit v3IlluminaCat#: FC-110–3001
Software, AlgorithmQiime2https://qiime2.org/V.2019.1
Software, AlgorithmDADA2 PackagePMID:27214047
Software, AlgorithmHuman Oral Microbiome Database (HOMD)Homd.org
PMID:30534599
v. 15.1
Software, AlgorithmR-studiohttps://rstudio.com/RRID:SCR_000432Version 3.5.3
Software, AlgorithmPhyloseqPMID:23630581RRID:SCR_013080
Software, AlgorithmClustvisPMID:25969447RRID:SCR_017133
Software, AlgorithmGgplot2https://www.springer.com/gp/book/9780387981413RRID:SCR_014601https://www.springer.com/gp/book/9780387981413
Software, AlgorithmAmpvis2http://dx.doi.org/10.1101/299537v1
Software, Algorithmveganhttps://cran.r-project.org, https://github.com/vegandevs/veganRRID:SCR_011950
Software, AlgorithmAde4https://www.jstatsoft.org/article/view/v086i01
Software, AlgorithmBioinformatic scripts and microbiome data used in analysisThis paperhttps://github.com/kkerns85/Rapamycin_rejuvenates_oral_health_in_aging_mice.git.
Software, AlgorithmR MarkdownThis paperhttps://rpubs.com/kkerns85/Rapamycin_Rmrkdown
Software, AlgorithmGraphpad PrismGraphpad (graphpad.com)RRID:SCR_002798Version 8.4

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