1. Presented by: Mari Springer AGE DEPENDENT LOSS OF MMP-3 IN HUTCHINSON- GILFORD PROGERIA SYNDROME

2.  Rare, premature aging disease  Progressive disease  Imbalance connective tissue  Characteristics  Short stature  Scleroderma-like skin  Progressive joint contracture  Atherosclerosis HUTCHINSON-GILFORD PROGERIA SYNDROME (HGPS)

3.  Premature death by heart attack, stroke, or atherosclerotic disease  Average age of 13  Progerin – altered version of lamin A protein  Point mutation  Chromosome 11  Depression of the enzyme MMP-3 contributes to HGPS HUTCHINSON-GILFORD PROGERIA SYNDROME (HGPS)

4.  Also known as lamin A/C  Protein encoded by LMNA gene  Functions  Aides in chromatic organization, DNA replication, transcription, and repair  Provides structure for the nuclear envelope  LMNA gene encodes for the protein prelamin A  Prelamin A has a farnesyl group attached to it’s end LMNA PROTEIN Normal processProgeria process Farnesyl group is removedFarnesyl group stays attached Prelamin A is codedProgerin is coded Nothing attached to nucleusProgerin attached to nucleus Normal nucleusAbnormal shaped nucleus

5.  Family of enzymes that degrade the extracellular matrix (ECM)  Maintain proper balance between ECM synthesis and degradation  Family includes MMP-1 through MMP-28  This paper focuses on MMP-2, -3, and -9  MMP-3 has the broadest substrate specificity  Degrade most of the basement membrane  Helps rebuild connective tissue MATRIX METALLOPROTEINASES (MMP)

6.  Is MMP-3 mRNA and MMP-3 protein regulation defective in HGPS patients?  Does the production of MMP-3 in HGPS cells change over time?  If changes are present, are they MMP-3 specific or general to the MMP family? GOALS

7.  Cell Lines  Obtained skin fibroblasts from the Progeria Research Foundation Cell and Tissue Bank and the Coriell Cell Repository  HGPS and non-HGPS lines  Ages 2, 3, 9, 10, and 13  Western Blot  To detect lamin A/C, prelamin A, and progerin  Real-Time Reverse Transcription PCR  To amplify genes  MMP-2, -3, and -9 and β-actin  MMP protein levels  MMP-2 and -9 used Gel Zymography  MMP-3 used Enzyme-Linked Immunosorbent Assay (ELISA) METHODS

8.  Statistical Methods  Linear Mixed Model  Used to show MMP mRNA levels relative to β-actin  Used to show MMP-3 protein levels  Pearson’s Correlation  Used to see relationship between number and MMP expression  Standard t-test  Used to show any other results METHODS

9.  Western Blot  HGPS lines – produced prelamin A, lamin A, lamin C, and progerin  Donor age-matched counterparts – produced prelamin A, lamin A, and lamin C RESULTS

10.  Reverse Transcription PCR  MMP-3 mRNA – 47-fold lower (p=.0107)  MMP-2 mRNA – 4.8-fold lower (p=.0275)  MMP-9 mRNA – not significantly different  Significant donor age- dependent decline in HGPS fibroblasts  MMP-3 p<.001  MMP-2 p<.003 RESULTS

11.  ELISA  MMP-3 protein levels reduced 10-fold in HGPS fibroblasts  More significant with increasing donor age  Gelatin Zymography  MMP-2 and -9 protein levels not significantly different RESULTS

12.  Is MMP-3 mRNA and MMP-3 protein regulation defective in HGPS patients?  Yes – reduced amounts in primary dermal fibroblasts  Does the production of MMP-3 in HGPS cells change over time?  Yes – significant decline in both mRNA and protein levels  Suggests a correlation with disease severity  Suggests altered balance in connective tissue remodeling  If changes are present, are they MMP-3 specific or general to the MMP family?  MMP-3 is specifically downregulated in HGPS DISCUSSION

13.  MMP-3 could be a potential biomarker to aide in the process of finding treatments and improving existing ones  What are other potential biomarkers for HGPS?  Are these helpful in other diseases affected by the MMP-3 enzyme RESEARCH PROPOSAL

14.  al., M. A. (2008). Phenotype and Course of Hutchinson-Gildord Progeria Syndrome. The New England Journal of Medicine, 592- 604.  Halaschek-Wiener, J., & Brooks-Wilson, A. (2007). Progeria of Stem Cells: Stem Cell Exhaustion in Hutchinson-Gildfor Progeria Syndrome. Journal of Gerontology, 3-8.  Harten, I. A., Zahr, R. S., Lemire, J. M., Machan, J. T., Moses, M. A., Doiron, R. J., et al. (2011). Age-Dependent Loss of MMP-3 in Hutchinson-Gilford Progeria Syndrome. Journal of Gerontology: Biological Sciences, 1201-1207.  Rastogi, R., & Mohan, S. C. (2010). Progeria Syndrome: A Case Report. Indian Journal of Orthopaedics, 1-9.  Tortora, G. J., Funke, B. R., & Case, C. L. (2010). Microbiology: An Introduction. San Francisco: Pearson Benjamin Cummings.  Wamer, H. R. (2008). Research on Hutchinson-Gilford Progeria Syndrome. Journal of Gerontology: Biological Sciences, 775-776. BIBLIOGRAPHY