Mitochondria morphology and DNA content upon sublethal exposure to beta-amyloid(1-42) peptide

Diana, Andrea and Šimić, Goran and Sinforiani, Elena and Orrů, Nicola and Pichiri, Giuseppina and Bono, Giorgio (2008) Mitochondria morphology and DNA content upon sublethal exposure to beta-amyloid(1-42) peptide. Collegium Antropologicum, 32 (Suppl.). pp. 51-58. ISSN 0350-6134

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Abstract

Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between amyloid beta peptides (AP) and mitochondrial dysfunction has been established in cellular models of AD using Abeta concentrations capable of triggering massive neuronal death. However, mitochondrial changes related to sublethal exposure to Abeta are less known. Here we show that subtoxic, 1 microM Abeta(1-42) exposure does not change the mitochondrial shape of living cells, as visualized upon the uptake of the non-potentiometric fluorescent probe Mitotracker Green and enhanced yellow fluorescent protein (EYFP)-tagged cytochrome c oxidase expression. Immunolocalization of oxidative adducts 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanine and 8-hydroxyguanosine demonstrates that one-micromolar concentration of Abeta(1-42) is also not sufficient to elicit dramatic qualitative changes in the RNA/DNA oxidative products. However, in comparison with controls, semi-quantitative analysis of the overall mitochondrial mass by integrated fluorescence intensity reveals an ongoing down-regulation in mitochondrial biosynthesis or, conversely, an enhanced autophagic demise of Abeta treated cells. Furthermore, a significant increase of the full-length mitochondrial DNA (mtDNA) from Abeta-treated versus control cells is found, as measured by long range polymerase chain reaction (PCR). Such up-regulation is accompanied by extensive fragmentation of the unamplified mtDNA, probably due to the detrimental effect of Abeta. We interpret these results as a sequence of compensatory responses induced by mtDNA damage, which are devoted to repression of oxidative burst. In conclusion, our findings suggest that early therapeutic interventions aimed at prevention of mitochondrial oxidative damage may delay AD progression and help in treating AD patients.

Abstract in Croatian

U mozgova osoba s Alzheimerovom bolešću (AD) vidi se veliki spektar mitohondrijalnih promjena kako na morfološkoj, tako i na genetskoj razini. U staničnim modelima AD pronađena je kauzalna povezanost između prisutnosti peptida beta-amiloida (Ab) i disfunkcije mitohondrija samo uz upotrebu koncentracija Ab koje su sposobne izazvati masivnu smrt neurona. No, mitohondrijalne promjene povezane sa subletalnom ekspozicijom Ab nisu tako dobro proučene. U ovom smo istraživanju pokazali da subtoksična ekspozicija s 1 mM Ab1–42 ne mijenja oblik mitohondrija stanica u kulturi, što je jasno vidljivo nakon vizualizacije mitohondrija pomoću na razlike u potencijalu neosjetljive fluorescentne probe Mitotracker Green i na temelju ekspresije citokrom c oksidaze označene modificiranim žutim fluorescentnim proteinom s poboljšanom emisijom (EYFP). Imunolokalizacija oksidativnih kompleksa 8-hidroksi-2’-deoksigvanozina, 8-hidroksigvanina i 8-hidroksigvanozina pokazala je da jednomolarna koncentracija Ab1–42 također nije dovoljna da bi izazvala dramatične kvalitativne promjene vizualiziranih oksidativnih produkata RNA i/ili DNA. Ipak, u usporedbi s kontrolama, semikvantitativnom analizom mitohondrijalne mase izmjerene iz intenziteta ukupne fluorescencije otkrili smo značajne razlike koje ukazuju da dolazi ili do sveukupnog smanjivanja biosinteze mitohondrija ili pak da se radi o pojačanoj autofagičkoj eliminaciji stanica tretiranih s Ab. Nadalje, upotrebom lančane reakcije polimeraze dugog odsječka (long range PCR) u stanica tretiranih s Ab pronašli smo značajan porast cijele mitohondrijalne DNA (mtDNA). Ovakva pojačana sinteze cijele mtDNA bila je popraćena izraženom fragmentacijom neamplificirane mtDNA, vjerojatno uslijed štetnog djelovanja Ab. Dobivene rezultate interpretirali smo kao niz kompenzatornih procesa izazvanih oštećenjem mtDNA, kojima je cilj zaustavljanje naglog porasta oksidacije. Zaključno, naši nalazi ukazuju da bi rane terapijske intervencije usmjerene na prevenciju oksidativnog oštećenja mitohondrija mogle odgoditi progresiju AD i pomoći u liječenju bolesnika s AD.

Item Type: Article
MeSH: Amyloid beta-Protein - toxicity ; DNA, Mitochondrial - drug effects - genetics ; Electron Transport Complex IV - genetics ; Mitochondria - drug effects - metabolism ; Oxidative Stress - drug effects ; Peptide Fragments - toxicity ; Aldehydes ; Animals ; Cell Line, Tumor ; Fluorescent Dyes ; Mice ; Transfection
Departments: Hrvatski institut za istraživanje mozga
Depositing User: Lea Škorić
Status: Published
Creators:
CreatorsEmail
Diana, AndreaUNSPECIFIED
Šimić, GoranUNSPECIFIED
Sinforiani, ElenaUNSPECIFIED
Orrů, NicolaUNSPECIFIED
Pichiri, GiuseppinaUNSPECIFIED
Bono, GiorgioUNSPECIFIED
Date: January 2008
Date Deposited: 16 Jun 2008
Last Modified: 23 Sep 2011 16:10
Subjects: /
Related URLs:
    URI: http://medlib.mef.hr/id/eprint/373

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