Department of Biology, Faculty of Exact and Natural Sciences,

1. The Influence of Brain Derived Protein Complex on the ProliferativeActivity of Hippocampal Progenitor Cells GiorgiMosidze, M.S. Department of Biology, Faculty of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia 2012

2. NEUROGENESIS ?!

3. Rat’s Hippocampus • 1-CA1; 2-CA2; 3-CA3fields; • 4-suprapiramidal and5-infrapiramidal blades of dentate gyrus. • 6-polymorphic cell layer(hilus) (7X3).

4. Progenitor cells of rats’ dental gyrus CA1 CA3 DG Sub granular Layer

5. Regulation of Neurogenesis

6. IGF I

7. ETPC- Endogenous Thermostable Protein Complex(Investigation was started by Prof. Tumanishvili) • Ethanol extraction according to the Bullough method (Bullough et al 1964) Note: Bullough studied endogenous growth factors and extracts of vertebrate epidermis contain an antimitotic chemical messenger, called the epidermal chalone, which appears to be of central importance in the control of epidermal mitosis. • The termostable complex was received by boiling at 100 C ; • Different sources of extraction: • Phylogeneticaly diverse species (Bacteria, ......human); • Different organs of one species (in our case rat)

8. Chemical Characterization Native PAGE Bovine albumin 66.00 kDa Fraction I – at molecular weight ranged between 45-66 kDa Egg albumin 45.00kd Fraction II – at molecular weight ranged between 12-17 kDa Horse myoglobin17.00kd CytochromeC 12.00kd Brain Heart

9. Chemical Characterization hydrophobic-interaction HPLC The same picture as at electrophoresis -two fractions in the extracts from different organs Fraction I – corresponds to the second peak (hydrophobic) Fraction II – corresponds to the first peak at the diagram (hydrophilic);

10. Influence on Transcriptional Activityin vitro test system of isolated nuclei Intensity of RNA synthesis % Intact nuclei Nuclei + ETPC

11. Inhibition of Mitotic Index • Control • Experiment Note: experiment means the effect of ETPC after 3 hours of injection

12. Due to inhibition of transcriptional activity, ETPC decreases the mitotic index .

13. Specificity The tissue and species specificity were studied both at the level of transcriptional and mitotic activity. The ETPC is tissue-specific, but it is not shown to be species - specific. Intensity of RNA synthesis % Control Heart Liver Brain

14. However, the tissue-specificity reveals only in case of terminally differentiated cells.

15. GOAL ! To study the influence of endogenous protein complex on the proliferative activity of newborn rats’ brain progenitor cells.

16. Materials and Methods • Materials: • Brain tissue of newborn (5-6 days) rats • Adult Rats’ brain for the ETPC extraction • Methods: • Extraction of protein complex according to the Bullough method (Bullough et al 1964). • Histochemical staining (hematoxilin/eosin) • Imunohistochemical staining ( against Ki 67)

17. G2 phase Mitosis

18. Changing of mitotic activity of dental gyrus cells, in 3 hours after ETPC injection. P<0.002

19. Mitotic figures of dentate gyrus of hippocampus B A 7X90

20. Changing of mitotic activity in CA1 and CA3 fields of hippocampus in 3 hours of ETPC injection. P>0.2 P>0.05

21. Protein complex derived from adult rats, decreases the mitotic activity of dentate gyrus of newborn rats’ hippocampus.

22. Changing of Ki 67 positive cells’ quantity in dentate gyrus, in three hours after protein complex injection P<0.001

23. Ki 67 positive cells of dentate gyrus of newborn rats’ hippocampus. 7X40 7X90

24. Influence of ETPC on the quantity of Ki-67 positive cell G2 The increasing of Ki-67 positive cell quantity possibly defined by retention of cells in G2 phase and by the entering of new pool of cell into the cell cycle M S G1 G0

25. Is this process reversible or not?

26. The reversed effect of ETPC Equal mitotic index in control and experiment at 5 hours after injection Indicates on the reversed effect of ETPC

27. What might be the prospective mechanism of restoration of mitotic index?

28. Conclusion • Protein complex derived from adult rats brain decreases the mitotic activity in homologies tissue in newborn rats. • This inhibitory effect prolongs only three hours and is reversible process. • The cells, which were impeded in G2 phase, maintain ability to enter into mitosis.

29. Acknowledgement G. Tumanishvili† D. Dzidziguri N. Giorgobiani I. Modebadze E. Bakuradze L. Rusishvili M. Rukhadze T. AslamaziShvili and all members of working group.

30. Thanks for your attention

31. Influence of ETPC on the quantity of Ki-67 positive cell The quantity of Ki-67 positive cells is increased by the effect of ETPC

32. EPTC in Kidney Tumor EPTC was extracted from transformed cells. The ETPC in tumor cells was not identified Normal tissue Transformed tissue

33. The influence of ETPC on the different type of transformed cells MDCK Jurkat Cell Erlichascitic carcinoma Note: Transcriptional activity 1. control; 2. experiment ( effect of EPTC on Isolated nuclei)

34. ETCP-Possible using in Cancer Therapy Two key points, absence of ETPC in transformed cells and inhibition effect on transcriptional and correspondently mitotic levels, give us possibility to think of this protein complex as a potential component of cancer therapy.

35. The future study The future study strategy should be focused on more deep investigation of protein features, mechanism of acting in cancer cells. It will be interesting for our group to check if this protein complex, works according the schema advised by our group above.

36. Asymmetric distribution of Ki 67 positive cells in various fields of hippocampus (intact rats)

37. Changing of Ki67 positive cells' quantity in CA1 and CA3 fields in three hours after protein complex injection