MicroRNA profiling of the XFE progeroid syndrome demonstrates similarities between progeroid and wild-type aged mice Lolita S Nidadavolu, BA;1,2 Laura J Niedernhofer, MD/PhD;3 Saleem A Khan, PhD1 1University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics; 2University of Pittsburgh Medical Scientist Training Program; 3Scripps Florida, Department of Metabolism and Aging

Disclosure Statement The research reported during this presentation was supported by a pilot grant from University of Pittsburgh Cancer Institute (UPCI) and T32 AG021885 (PI: Studenski) . The investigators retained full independence in the conduct of this research

Cellular senescence can be induced by DNA damage Cellular senescence is another cellular outcome of aging. It is a state of cell cycle arrest, yet, the cell is non-apoptotic. Cells can undergo senescence via many pathways: Telomere shortening, in response to oncogenic stimuli. Stress that senescent cells are not metabolically inactive. In fact, they have recently shown to upregulate pro-inflammatory markers, such as IL-6 and IL-8 (Senescence Associated Secretory Phenotype) IL-6 IL-8 p16 J Campisi Nature Reviews Mol Cell Biol 8, 729- 740 (2007)

XFE Progeroid Syndrome and ERCC1-deficient Mice Age 8 Age 16 Xeroderma Pigmentosum F-Ercc1 (XFE) Progeroid Syndrome: Severe rapid aging phenotype Dwarfism, vision loss, cachexia Ercc1-/- and Ercc1-/Δ mice: Mouse model of XFE Progeroid Syndrome Lifespan: Ercc1-/- = 4 weeks; Ercc1-/Δ = 28 weeks DNA repair defect in the repair of: Bulky lesions, Double Strand Breaks, Interstrand Cross-Links The mouse model which most closely models the symptoms exhibited by the patient with XFE Syndrome is Ercc1-/- mice. These mice have severe growth restriction starting from 2 weeks after birth. They also exhibit the same liver and kidney abnormalities as well as other signs of aging like bone marrow abnormalities, and neurologic degeneration including ataxia. These mice also have much shorter lifespan than WT mice, with death by 4 weeks. Previous papers have shown that these mice also exhibit severe sensitivity to oxidative stress and early senescence. UV irradiation looking at NER pathway Mitomycin C – DNA crosslinker L Niedernhofer Nature 444, 1015-1071 (2006)

What do miRNAs do? Post-transcriptional regulation of genes miRNA Full complementarity: Degrades mRNA Imperfect pairing: Suppresses protein translation miRNA Target protein MicroRNAs (miRNAs) are ~22 nt long single-stranded RNAs that negatively regulate their target genes, dysregulating cellular pathways. Recently, differential expression of miRNAs has been observed in a variety of human diseases, especially cancers. MicroRNA Biogenesis overview: After transcription miRNAs fold into primary miRNA dsDNA structures of varying length, and are then processed into precursor miRNAs that are 70 nt long. After nuclear export, pre-miRNAs are cleaved by the Dicer enzyme and yield 20 nt duplexes. After this processing one strand is incorporated into a RNA-induced silencing complex (RISC) which contains proteins in the Argonaute family. The RISC targets the 3’UTR of mRNAs and can either completely degrate or translationally repress targets.

Hypothesis We hypothesize that analyzing miRNA expression in Ercc1-deficient cells and tissues can identify miRNAs dysregulated in normal aging These miRNAs can be further studied to identify pathways and mechanisms that regulate cellular senescence and aging

Different miRNAs are expressed in Ercc1-/- MEFs compared to WT MEFs microRNA Fold-change p-value miR-301a -2.24 0.007 miR-543 -2.60 0.023 miR-326 -3.05 0.029 miR-455* -3.23 0.020 miR-872 -6.72 0.044 miR-497 -6.87 0.002 microRNA Fold-change p-value miR-467a 2.19 0.018 Differentially expressed miRNAs in late passage Ercc1-/- MEFs compared to WT MEFs in 20% O2. Differentially expressed miRNAs in late passage Ercc1-/- MEFs compared to WT MEFs in 3% O2.

Different miRNAs are expressed in senescent versus pre-senescent Ercc1-/- MEFs microRNA Fold-change p-value miR-671-5p 14.31 0.025 miR-29b* -2.15 0.003 miR-1892 12.73 0.041 miR-449a -2.24 0.043 miR-483 12.60 0.020 miR-455* -2.77 0.004 miR-1894-3p 8.46 0.050 miR-340-3p -2.96 0.036 miR-1895 7.28 0.039 miR-362-5p -3.56 0.038 miR-680 6.54 0.023 miR-675-3p -3.94 miR-721 6.43 0.029 miR-466a-3p -3.95 0.037 miR-129-5p 5.06 0.046 miR-128 -4.41 0.047 miR-1906 3.82 0.006 miR-497 -5.16 0.048 miR-222 3.73 0.034 miR-362-3p -5.23 0.012 miR-320 3.62 0.010 miR-192 -5.61 miR-290-5p 3.41 0.002 miR-496 -5.79 0.044 miR-22 3.27 miR-543 -6.81 miR-877 2.86 miR-30e* -8.15 0.016 microRNA Fold-change p-value miR-382* -10.07 0.029 miR-337-3p -11.34 0.049 miR-450b-3p -12.44 0.014 miR-872 -14.71 0.021 miR-369-5p -15.11 0.024 miR-380-3p -15.73 0.048 miR-154* -31.57 0.019 miR-10b -32.48 0.041

Confirmation of miRNA expression by qRT-PCR Comparison of senescent versus pre-senescent Ercc1-/- MEFs

A subset of miRNAs are downregulated in progeroid (Ercc1-/Δ) and WT old livers After validating miRNAs in MEFs, looked at liver tissues. The livers of Ercc1 mice are severely affected by the mutation, and we wanted to see if the miRNAs that we found from the array were dysregulated in livers. WT mice were grouped with Ercc1 hets, because they are relatively unaffected by the loss of one allele. However, for the 8 miRNAs listed here, there were similar down-regulation trends in the hypomorphic mice and in the WT old mice livers. Ages (young = 20 weeks, old = 30 months)

A subset of miRNAs are downregulated in progeroid (Ercc1-/Δ) and WT old kidneys

Summary Differential miRNA expression in: Ercc1-/- vs. WT MEFs Early passage vs. Late passage Ercc1-/- MEFs (3% O2) More miRNAs downregulated with senescence 8 miRNAs with same expression patterns in livers WT young and Ercc1+/Δ similar expression WT old and Ercc1-/Δ similar expression 3 miRNAs with similar expression in kidneys

Future Directions Functional studies (Over-express miRNAs in WT MEFs) Changes to senescence, proliferation, apoptosis miRNA Target Analysis Luciferase 3’UTR Assays to confirm miRNA targeting Promoter studies Bisulfite sequencing (CpG islands upstream) TF binding site analysis (NF-κB is increased in progeroid and normal aged mice)

Acknowledgements Saleem Khan, PhD Laura Niedernhofer, MD/PhD Cecilia Munko Parvez Akhtar Laura Niedernhofer, MD/PhD Siobhan Gregg Andria Robinson Thesis Committee: Abbe de Vallejo, PhD Ferruccio Galbiatti, PhD Paul Robbins, PhD This work was supported by a pilot grant from the University of Pittsburgh Cancer Institute and T32 AG021885 (PI: Studenski)

MicroRNA profiling of the XFE progeroid syndrome demonstrates similarities between progeroid and wild-type aged mice Lolita S Nidadavolu, BA,1,2 Laura J Niedernhofer, MD/PhD,3 Saleem A Khan, PhD1 1University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics; 2University of Pittsburgh Medical Scientist Training Program; 3Scripps Florida, Department of Metabolism and Aging

microRNAs and senescence Recent review showing how different miRNAs can post-transcriptionally regulate both senescence and cannonical aging pathways. L.-H. Chen Ageing Res. Rev. (2010)

Nucleotide Excision Repair Nucleotide Excision Repair is involved in removing bulky, helix distorting lesions. Two types of NER: Transcription-coupled and global genome. TC occurs on the transcribed strand and comes into play when there are lesions that block RNA transcription. Key protein in this pathway is XPF-ERCC1 heterodimer, which is a 5’endonuclease. It’s involved in the actual excision step of NER. Essential to have both XPF and Ercc1 for the complex to be stable. XP – UV sensitivities, skin cancer. defects in global NER CS – premature aging symptoms (wasting and neurologic problems), defects in TC-NER M Fousteri Cell Research 18, 73–84 (2008)

ERCC1 Mutant Mice are sensitive to DNA damage The mouse model which most closely models the symptoms exhibited by the patient with XFE Syndrome is Ercc1-/- mice. These mice have severe growth restriction starting from 2 weeks after birth. They also exhibit the same liver and kidney abnormalities as well as other signs of aging like bone marrow abnormalities, and neurologic degeneration including ataxia. These mice also have much shorter lifespan than WT mice, with death by 4 weeks. Previous papers have shown that these mice also exhibit severe sensitivity to oxidative stress and early senescence. UV irradiation looking at NER pathway Mitomycin C – DNA crosslinker L Niedernhofer Nature 444, 1015-1071 (2006)

Dicer is downregulated in senescence and aging