1. 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

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

3. 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, (2007)

4. 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, (2006)

5. 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.

6. 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

7. 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.

8. 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 p
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

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

10. 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)

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

12. 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

13. 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)

14. 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 AG (PI: Studenski)

15. 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

16. 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)

17. 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)

18. 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, (2006)

19. Dicer is downregulated in senescence and aging