This document summarizes a student research project exploring the regulation of NRD-convertase expression by retinoic acid. The student aims to investigate how NRD-convertase levels in SK-N-BE cells are modulated by retinoic acid and tamibarotene at different time points and concentrations. The methods section describes setting up cell cultures, drug treatments, and using real-time PCR to measure NRD-convertase and actin levels. Preliminary results are shown in figures and tables. The conclusion is that retinoic acid plays a role in regulating NRD-convertase and may be important in brain disease pathogenesis, but more samples are needed to validate these results.

1. EXPLORING THE REGULATION OF NRD-CONVERTASE EXPRESSION BY RETINOIC ACID KRISHNA MOORTHY SUPERVISOR: DR. ADRIAN PIEROTTI

2. INTRODUCTION NRD-CONVERTASE (NRDc) is a cytosolic and cell-surface metallopeptidase enzyme. It is classified under Clan ME in Family M16, Subfamily A, M16.005 (merops). This enzyme is present in abundant amount in endocrine tissues, in rat testis at late stages of spermatogenesis and brain cortex.

3. INTRODUCTION CONTINUED…. The 90 amino acid acidic domain inserted close to NRDc active site differentiates it from other members of the M16 family of metalloendopeptidases. NRDc acts by cleaving arginine residues on N-terminus of basic amino acid or lysine in basic pairs.

4. IMPORTANCE OF ENZYME Role in Alzheimer Disease, Brain Diseases, Down Syndrome, Leukemia Neoplasms and Neuroblastoma

6. BE(2)-NA cell lines showed 9-fold increase in the expression of NRD Convertase within few hours to a day treatment.

9. Figure 1. Samples of SK-N-BE cells of Actin and NRDc on gel electrophoresis

11. Figure 2. Restriction digest of Actin and NRDc on gel electrophoresis

12. STD OF ACTIN CONCENTRATION OF ACTIN

13. STD OF NRD CONCENTRATION OF NRD

14. ACTIN IN COMBO WITH NRD CONCENTRATION OF ACTIN

15. NRD in combo CONCENTRATION OF NRD

16. CELL TISSUE CULTURE TREATMENTS 1µM TAMBIROTENE 1µM RETINOIC ACID 5µM RETINOIC ACID TIMES 6 HOURS 24 HOURS 48 HOURS MEASURE Ct NRD Ct ACT in same tube Calculate ∆Ct = CtNRD-CtACT Calculate ∆∆Ct = ∆CtControl-∆CtTreatment ∆∆Ct = 1 = 2 fold change

17. ∆∆Ct of NRDc level

18. FOLD CHANGE OF NRDc LEVEL AFTER DRUG TREATMENT FOLD CHANGE OF NRDc LEVEL AFTER

19. BAR CHART REPRESENTATION OF FOLD CHANGE OF NRDc LEVEL

20. Conclusion: As presence of retinoic acid causes changes in expression of NRD levels in human SK B NE cells, it can be concluded that retinoic acid plays an important role in pathogenesis of brain diseases. The results is not similar with previous works. This may be due to small number of samples treated. To get more accurate result this experiment must be repeated with more number of treatment.