Giorgi Mosidze, M.S.

The Influence of Brain Derived Protein Complex on the Proliferative

Activity of Hippocampal Progenitor Cells

Department of Biology, Faculty of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State University,

Tbilisi, Georgia

2012

NEUROGENESIS ?!

Rat’s Hippocampus

1-CA1; 2-CA2; 3-CA3 fields;

4-suprapiramidal and 5-infrapiramidal blades of dentate gyrus.

6-polymorphic cell layer(hilus)(7X3).

Progenitor cells of rats’ dental gyrus

CA1

CA3

DG

Sub granular Layer

Regulation of Neurogenesis

IGF I

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)

Chemical Characterization Native PAGE

Bovine albumin 66.00 kDa

Egg albumin 45.00kd

Horse myoglobin 17.00kd

Cytochrome C 12.00kd

Brain Heart

Fraction I – at molecular weight ranged between 45-66 kDa

Fraction II – at molecular weight ranged between 12-17 kDa

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);

Influence on Transcriptional Activity

in vitro test system of isolated nuclei

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Inte

nsity

of R

NA

synt

hesi

s %

1. Intact nuclei2. Nuclei + ETPC

Inhibition of Mitotic Index

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Mito

tic I

ndex

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1. Control 2. ExperimentNote: experiment means the effect of ETPC after 3 hours of injection

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

Specificity

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Inte

nsity

of R

NA

synt

hesi

s %

Control Heart Liver Brain

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.

Con-trol

Pig Mouce water cavy

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100120

Inte

nsity

of R

NA

synt

hesi

s %

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

GOAL !

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

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)

G2 phase

Mit

osis

Changing of mitotic activity of dental gyrus cells, in 3 hours after ETPC injection.

Control Experimental0

0.51

1.52

2.53

3.54

4.55

Mito

tic in

dex

‰

P<0.002

Mitotic figures of dentate gyrus of hippocampus

7X90

A B

Changing of mitotic activity in CA1 and CA3 fields of hippocampus in 3 hours of ETPC injection.

Cont

rol

Expe

rimen

tal012345

CA 1

Mito

tic

inde

x ‰

Control Experimental0

0.51

1.52

2.53

3.54

4.55

CA3

Mito

tic in

dex

‰P>0.05 P>0.2

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

Changing of Ki 67 positive cells’ quantity in dentate gyrus, in three hours after protein complex injection

Control Experimental0

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Ki 6

7 p

ositi

ve c

ells

‰

P<0.001

Ki 67 positive cells of dentate gyrus of newborn rats’ hippocampus.

7X40 7X90

Influence of ETPC on the quantity of Ki-67 positive cell

G2

M

G1

S

G0

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

Is this process reversible or not?

The reversed effect of ETPC

3 5 70

0.51

1.52

2.53

3.54

4.5

controlexperiment

Hour

Mito

tic in

dex

‰

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

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

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.

Conclusion

Acknowledgement

G. Tumanishvili †

D. Dzidziguri

N. Giorgobiani

I. Modebadze

E. Bakuradze

L. Rusishvili

M. Rukhadze

T. AslamaziShvili and all members of working group.

Thanks for your attention

Influence of ETPC on the quantity of Ki-67 positive cell

Control experiment05

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Ki -

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The quantity of Ki-67 positive cells is increased by the effect of ETPC

EPTC in Kidney Tumor

EPTC was extracted from transformed cells. The ETPC in tumor cells was not identified

Normal tissue

Transformed tissue

The influence of ETPC on the different type of transformed cells

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MD

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Note: Transcriptional activity 1. control; 2. experiment ( effect of EPTC on Isolated nuclei)

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.

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.

The future study

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

DG CA1 CA305

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Ki 6

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

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