Apoptóza ako proces adaptácie (?)Three main death processes have been characterized: the autophagy, which is an execution of unwanted parts of the cell, the ap\൯ptosis, which affects

$Page 1: Apoptóza ako proces adaptácie (?)Three main death processes have been characterized: the autophagy, which is an execution of unwanted parts of the cell, the ap\൯ptosis, which affects$
Apoptóza ako proces adaptácie (?)

Pavel BABÁL

Apoptóza = programovaná smrť bunky ako proces adaptácie

Stimulácia bunkovej smrti spustením priepustnosti mitochondriálnej membrány - mitochondrial permeability transition (MPT):

*Otvorenie MPT pórov je znakom smrti bunky a často sa považuje za „moment bez návratu“

*Ktorý typ bunkovej smrti sa uplatní závisí od počtu postihnutých mitochondrií.

*Mitochondrie v blízkosti ER/SR skôr otvárajú svoje póry, osobitne pri oxidatívnom strese.

Typy bunkovej smrti a úloha mitochondrií na ich spustení (Lemasters et al, Biochimica et Biophysica Acta, 1998, 1366, 177-196)

odpo

veď

Prezentujúci

Poznámky prezentácie

Three main death processes have been characterized: the autophagy, which is an execution of unwanted parts of the cell, the apoptosis, which affects single cells, and the necrosis, which affects group of cells. A universe fature that precedes the three types of CD is the opening of the mitochondrial permeability transition pore (MPTP). ATP depletion, overload by Ca2+ or block of the proteins stabilizing the mitochondrial membrane have been considered as usual preconditions preceding the MPTP opening. Which type of death process will develop depends on the number of affected mitochondria.

Apoptóza = programovaná smrť smrť bunky NEKRÓZA APOPTÓZA - postupný úplný rozpad - scvrknutie bunky, bunkových štruktúr po kondenzácia chromatínu a smrti bunky cytoplazmy, fragmentácia (dôsledok nezvratného bunky, fagocytóza. poškodenia bunky)

Morfologické prejavy

Biochemické zmeny

NEKRÓZA APOPTÓZA ↑ priepustnosť plazmatickej - aktívny metabolický proces membrány - zmeny povrchovej membrány ↓ energetických zásob – opuch - zmeny v cytoplazme (mikro- ↑ intracelulárne Ca++ filamenty) … aktivácia fosfolipáz - zmeny proteosyntézy …rozpad membránových jednotiek - štiepenie DNA (bunková membrána, mitochondrie, - aktivácia kaspáz lysozómy) - formovanie apoptózových teliesok

Prezentujúci


This work concerns the possible role of AIF in the myocyte dedifferentiation after T. Sp. occupation and its relationhip with Bax, Bcl-2 and caspase-3 in the conditions of asynchronous invasion by means of routine histochemical methods.

Prezentujúci



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Bunková membrána

AIF

Cytoplazma Smrtné signály

Bax

Bcl-2

cytochróm c

M RIP

TNF TNF TNF

FADD

TNFR1

DD

Aktivácia kaspáz

Death inducing signaling complex (DISC)

TRADD

prokaspáza-3 kaspáza-9

prokaspáza-9

cytochróm c

Apaf-1

Bax

N

DNA fragmentácia

N kaspáza-3

kaspáza-8, 10

prokaspáza-3

Prezentujúci


There are two different mechanisms by which a cell commits suicide. The first one is generated by signals arising within the cell-this is the intrinsic or mitochondrial pathway. The another is triggered by death activators (TNF, FAS-ligand and lymphotoxin-they all are cytokines) binding to receptors on the cell surface-this is the extrinsic or death receptor pathway. The final step of both mechanisms is the caspase-3 activation, which is one of the mains executioners of apoptosis. In some cells, like the neurons, a direct caspase-independent pathway is involved through AIF. Under nuclear stimulation it relocates from the MX to the nucleus, where it causes a large DNA fragmentation. The AIF in cytoplasm may induce cytochrom-c release and subsequent caspase-3 activation.. This work concerns the possible role of AIF in the myocyte dedifferentiation after T. Sp. occupation and its relationhip with Bax, Bcl-2 and caspase-3 in the conditions of asynchronous invasion .

Trichinella spiralis parazitický červ

Prezentujúci


Trichinellosis results from infection by a parasitic nematode belonging to the genus Trichinella. The acute stage of the infection occurs in the intestine, where the trichinelas reproduce. The chronic stage occurs only in the striated muscles, where the newborn larvae accommodate. After penetrating the myocyte, they induce dramatically changes in the cell, which result in unique structure called nurse cell.

Intestinálne štádium životného cyklu Trichinely


HOH C

HO

N H C

H 3 C

2

OH

HO

O

1

2

3

4

COOH

O O

TML molecule

5

6 7

8 9

Tritrichomonas mobilensis lektín

Sialová kyselina


Adhézia Tritrichomonas mobilensis na črevný epitel → T. mobilensis lektín - kyselina sialová ? Využíva Trichinella spiralis sacharidovú väzbu na črevný epitel v iniciálnej fáze infekcie ?



? Využíva Trichinella spiralis sacharidovú väzbu na črevný epitel v iniciálnej fáze infekcie ? : Nevyužíva väzbu na sialovú kyselinu ani iný sacharid .....

Epitelové bunky obaľujúce parazita bez znakov nekrózy alebo apoptózy

Aktivácia faktorov apoptózy v bunkách okolo parazita:

BAX Bcl-2

AIF Kaspáza 3

ZÁVER I - Trichinella spiralis nevyužíva sacharidmi sprostredkovanú adhéziu na slizničný epitel - Trichinella spiralis neaktivuje proces odumierania v prevŕtaných bunkách črevnej sliznice

Prezentujúci


Trichinellosis results from infection by a parasitic nematode belonging to the genus Trichinella. The acute stage of the infection occurs in the intestine, where the trichinelas reproduce. The chronic stage occurs only in the striated muscles, where the newborn larvae accommodate. After penetrating the myocyte, they induce dramatically changes in the cell, which result in unique structure called nurse cell.

Morfológia živnej bunky Trichinella spiralis

Trichinella L1-larva Cytoplazma živnej bunky okolo parazita

Jadrá živnej bunky

Jadrá živnej bunky

Jadro satelitnej bunky

Kolagénová kapsula

Nepostihnuté myocyty

Prezentujúci


This nurse cell is derived from a portion of striated muscle cell after dedifferentiation. The molecular mechanisms at work that result in this unique relationship still have plenty of gaps. However, the mechanisms of apoptosis are likely to occur.

? úloha mechanizmov apoptózy v procese formovania živnej svalovej bunky a účasť faktorov apoptózy Bax, Bcl-2, AIF, PARP-1 a kaspázy-3 v podmienkach synchrónnej invázie Trichinella spiralis (ISS03).

BALB/C myši, samci 6-8 týždňov staré boli inokulované 500 infekčnými Trichinella spiralis larvami per os. V anestéze boli myši utratené - 0, 10, 14 and 45 dní po inokulácii (d.p.i.). na

histologické a imunohistochemické vyhodnotenie.

Prezentujúci



Vzorky svalu v procese formovania živnej bunky 10 d.p.i.

H&E Bax AIF

Bcl-2 Kaspáza-3

Prezentujúci


At day 10 p.i. we have low Bax and Bcl-2 expression at the sites of occupation, very strong AIF expression I the cytoplasm and the nuclei and the caspase-3 expression was limited at several spots only at one occupied area.

M Bcl-2

Cytochrom c

Bax

Bax Cytochrom c

Kaspáza-3

Bax 10 d.p.i. Bcl-2

1 µm

Matsuo et al., Parasitology, 2000, 121, 203-210

1 µm 1 µm

Matsuo et al., Parasitology, 2000, 121, 203-210

Prezentujúci


Trichinella enters the myocyte as simply perforate the sarcolemma. Disturbance of the cell membrane is one of the most common stimuli for up-regulation of Bax, that subsequently triggers the mitochondrial pathway of apoptosis. At the very beginning of the muscle occupation, however, the expression both of Bax and Bcl-2 in the affected areas was weak. To explain this result I accepted already reported assumption that at this moment the mitochondria swell and are probably destroyed by the lysosomes. Later new mitochondria appear, which are smaller.

Bax, 10 d.p.i.

Kaspáza-3, 10 d.p.i. AIF, 10 d.p.i.

M Bcl-2

Cytochrom c

N

Prezentujúci


In the process of apoposis by the caspase-dependent pathway, the mitochondrial membrane permeability disturbance is crucial for caspase-3 activation. Simultaneously, this may lead also to release of AIF within the cytoplasm. At the beginning of the invasion the decrease of Bax expression in the cytoplasm is accompanied with very low caspase-3 detection, only at one site of occupation. However, the same area shows very strong AIF expression in the cytoplasm and also inside of the enlarged nuclei. That why, this pathway doesn't seem to be very likely.

M

Cytochrome c

N

Bax, 10 d.p.i.

AIF, 10 d.p.i. H&E, 10 d.p.i.

Kaspáza-3, 10 d.p.i.

DNA fragmentacia N

PARP-1 poly-(ADP-ribose)-polymerase-1

M

Cytochrom c

N PARP-1

Prezentujúci


Therefore, in the conditions of low Bax and caspase-3 expression and strong AIF expression in the cytoplasm and also the nuclei at day 10 p.i. we suggest that AIF-mediated and caspase-independent and Bax independent signaling is involved in the apoptosis activation in the area occupied by Trichinella spiralis. A possible reason for relocation of AIF from the mitochondria to the nucleus is over expression of PARP-1-in this case the nucleus transmits a signal to the mitochondria. In the case of Trichinella accommodation, the infected cell undergoes dramatic changes leading to dedifferentiation in a nurse cell. These changes require intense nuclear processes illustrated by the nuclear hypertrophy so that the over-expression of PARP-1 is possible.


PARP-1 AIF Kaspáza-3

H&E Bax Bcl-2

Prezentujúci


14 d.p.i. we found that Bax and Bcl-2 are presented much more in the enlarged nuclei. Since hey both lack a nuclear translocation sequence it is not clear how they entered the nuclei or what is their function there. At this moment the caspase-3 also was presented in the cytoplasm and much more into the nuclei.

AIF, 10 d.p.i.



N

M

Cytochróm c

Prezentujúci


In the absence of caspase-3 AIF is required and sufficient to induce apoptosis. On the other hand this AIF, which is in the cytoplasm is able to provoke cytochrom-c release from the mitochondria, which could explain our findings of the absence of caspase-3 at day 10 p.i. and its appearance on day 14 p.i.


H&E Bax Bcl-2

PARP-1 AIF Kaspáza-3

Prezentujúci


At 45 d.p.i the occupied portion of he myocyte is already dedifferentiated toward a nurse cell, the Trichinella is accommodated inside, and the capsule formation around the nurse cell is completed. The staining with Bax and Bcl-2 is relatively weak, but AIF and capase-3 is missing. This means that at the beginning of the accommodation of Trichinella the mechanism of apoptosis are induced simply for to clean the space for Trichinella and to trigger the dedifferentiation, but after the nurse cell formation the apoptosis turns off.

Caspase-3, 14 d.p.i. AIF, 10 d.p.i.

Naša hypothesiséza – uplatňuje sa spätnoväzobná regulácia faktorov apoptózy?

Mitochondrie Jadro

+

PARP-1

AIF

Kaspáza-3

10-14 d.p.i.

Prezentujúci


If we accept that over-expression of PARP-1 is responsible for the induction of AIF relocation to the nuclei and AIF in the cytoplasm induces releasing of cytochrom c with subsequent caspase-3 activation isn't is possible caspase-3 to move into the nuclei namely to cleave PARP-1? PARP-1 is well documented nuclear substrate for the caspase-3. If so, this will stop the inducing signal and AIF release from the mitochondria, and also the AIF mediated cytochom c release. This is just a hypothesis, but in fact both AIF and caspase-3 are missing in the nurse cell and this complex remains viable about 15 years.

AIF, 45 d.p.i. Caspase-3, 45 d.p.i.

Mitochondrie Jadro

+

PARP-1

AIF

Kaspáza-3

45…….d.p.i.

Záver Relokácia AIF je pravdepodobne prvou udalosťou na začiatku procesu udomácnenia Trichinella spiralis v myocyte.

Zvýšená expresia niektorých faktorov apoptózy sa uplatňuje skôr v mechanizmoch adaptácie okupovanej bunky ako v procese vedúcom k smrti.

Normálne priečne pruhované svalové bunky

10 d.p.i. s T. spiralis

14 d.p.i. s T. spiralis 45 d.p.i. – komplex živnej bunky a T. spiralis

? Využitie získaných poznatkov... Trichinella spiralis vylučuje určité produkty, ktoré vstupujú do procesu regulácie bunkového cyklu – najprv stimuluje, potom inhibuje faktory apoptózy. Stimulácia apoptózy: - nádorové procesy - autoimunitné choroby - tlmenie nadmernej regenerácie/reparácie Inhibovanie apoptózy: - podporenie reparačných procesov - podporenie regeneračných procesov

R. Milcheva P. Janega A. Janegová L. Feketeová S. Petkova Z. Hurnikova M. Klincová I. Uhnavá

Documents

Apoptóza ako proces adaptácie (?)Three main death processes have been characterized: the autophagy, which is an execution of unwanted parts of the cell, the ap\൯ptosis, which affects