RIGO-Kunešová Gabriela

APLIKACE METOD TESTOVÁNÍ KOGNITIVNÍCH FUNKCÍ
U LABORATORNÍCH ZVÍŘAT
Rigorózní práce
Mgr. Gabriela Kunešová, Ph.D.
Přírodovědecká fakulta
Univerzity Palackého v Olomouci
Studijní program: Biologie
Obor: Zoologie
2012
1
ÚVOD
1. VLASTNOSTI LÁTEK S ÚČINKEM V CNS
Na CNS mohou účinkovat jen látky se schopností průniku přes hemato
encefalickou bariéru. Děje se tak buď specifickým transportním
mechanismem (přenašeče), nebo nespecifickým transportním mechanismem.
O způsobu transportu rozhodují fyzikálně-chemické vlastnosti látek, jako jsou
např. velikost molekuly, náboj, lipofilita. Ostatní látky jsou účinné jen tehdy,
jsou-li injikovány přímo do mozkomíšního moku. Hematoencefalická bariéra
je méně účinná v oblasti hypothalamu a oblastech, které jsou kolem třetí a
čtvrté komory. Její propustnost zvyšují některé chorobné stavy a zvýšená
tělesná teplota.
Účinek látek může být specifický (známý mechanismus účinku, specifické
receptory, vedou k účinku v koncentracích nižších, než jsou nutné pro látky
nespecificky působící) nebo nespecifický (svými f-ch vlastnostmi ovlivňují
membrány neuronů a mění tím průnik iontů membránami). Podle polarity
rozlišujeme účinek depresivní (stabilizace neuronální membrány >>> snížené
uvolňování transmiterů a odpovědi postsynaptických struktur) a stimulační
(blokáda inhibice nebo přímý excitační účinek).
Látky působící na CNS ovlivňují především úroveň vědomí, afektivity a
psychické integrace, bdění, chování, motoriky, vnímání bolesti apod.
U všech látek s centrálními účinky je nutné počítat s možností ovlivnění
chování, i když tato účinky jsou malé nebo nepozorovatelné za běžných
okolností.
2. LÁTKY S ÚČINKEM V CNS
a. Kognitiva (zlepšují vnímání, učení a paměť, působí především na mozkovou
kůru a limbický systém)
b. Anxiolytika (tlumí strach a úzkost)
c. Návykové látky (Syndrom závislosti - dán skupinou fyziologických,
behaviorálních a kognitivních jevů, přičemž užívání nějaké látky dává daný
jedinec mnohem větší přednost před jiným jednáním, které kdysi činil)
d. Hypnotika (navozují útlum CNS, mohou mít i účinky anxiolytické a
myorelaxační)
e. Psychofarmaka (mění duševní stav jedince, čtyři parametry psychiky: bdění a
vědomí, afektivita, psychická integrace, paměť)
f. Celková anestetika
g. Neuroleptika
h. Antiparkinsonika a antispastika
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3. KOGNITIVA
NOOTROPNÍ LÁTKY
zlepšují metabolismus a prokrvení CNS
- piracetam (klasické nootropní látky)
- námelové alkaloidy (látky s nootropní komponentou účinku)
OSTATNÍ KOGNITIVA
zvyšují hladinu acetylcholinu v mozku a napomáhají komunikaci mezi neurony
- inhibitory acetylcholiesterázy (takrin, galantamin)
- neuropeptidy (vazopresin)
- estrogeny
- antioxidancia (selen, karotenoidy)
- látky s nejasným mechanismem účinku (acetyl-l-karnitin)
4. KLASIFIKACE UČENÍ PRO PRAKTICKÉ TESTOVÁNÍ
Z FARMAKOLOGICKO-MEDICÍNSKÉHO HLEDISKA
KOGNITIV
a. klasické podmiňování:
i. nejjednodušší stupeň testování vyšší nervové činnosti
ii. není třeba funkčně dotvořené mozkové kůry
iii. široká škála živočišných druhů (i bezobratlých)
iv. nepodmíněný podnět >>> nepodmíněná (vrozená reakce)
nepodmíněný podnět + indiferentní podnět >>> reakce indiferentní
podnět >>> podmíněný podnět
v. podmíněný podnět >>> podmíněná reakce
b. podmíněné vyhýbání se (Shuttle box)
c. operantní (instrumentální) učení: zákon účinku - účinek správné asociace
podnětu s reakcí přináší úspěch; pokus a omyl s náhodným úspěchem (zvíře
má tendenci opakovat komplex pohybů, které vedou k cíli) (operantní box)
d. diskriminační učení (simultánní, sukcesivní) (jednoduché T bludiště)
e. prostorové učení a koncepce kognitivní mapy: živočich se učí spojovat
předměty a jevy vnějšího prostředí do komplexních souvislostí a prostorových
vztahů, vytváří si kognitivní mapu (Morrisovo vodní bludiště, komplexní Tbludiště, radiální bludiště) (1)
3
5. APLIKACE
METOD
TESTOVÁNÍ
KOGNITIVNÍCH
PROCESŮ
LABORATORNÍCH ZVÍŘAT NA KATEDŘE TOXIKOLOGIE FAKULTY
VOJENSKÉHO ZDRAVOTNICTVÍ V HRADCI KRÁLOVÉ
Výzkum účinku látek na chování a kognitivní schopnosti zvířat se od druhé poloviny minulého
století rozvíjel zejména v souvislosti potřeby získat podrobnější informace o látkách
využívaných globálně jako chemické bojové látky nebo prostředky teroristických útoků a v
druhé fázi i jejich antidot. Nejčastěji používané (protože nejsnáze a nejlevněji dostupné,
s rychlým nástupem projevů otravy a letálním účinkem) jsou látky ze skupiny organofosfátů.
Působí jako inhibitory acetylcholinesterázy, jejich intoxikace se projevuje muskarinovými,
nikotinovými a centrálními příznaky. Doložené záznamy případů, kdy byli lidé ovlivnění
nízkými dávkami chemických bojových látek, popisují fakt, že přestože se u zasažených
jedinců vůbec neprojevily klinické příznaky, došlo k narušení psychických, kognitivních a
emocionálních procesů. (2). Toto zjištění vedlo k výzkumu a vývoji takových antidot, která by
v případě intoxikace nejen potlačila fyzické účinky chemických bojových látek, ale navíc
zkvalitnila následný život intoxikovaných lidí ve smyslu reparace intelektuálněpsychologických schopností. Vedle výzkumu vojensky zajímavých látek se tým pracovníků
katedry podílí i na civilních projektech vývoji léčiv působících jako kognitiva. V poslední době
byla pozornost zaměřena na problematiku Alzheimerovy choroby (AD). Tato nemoc je
důsledkem narušení správné funkce stejného nervového systému, tedy cholinergního, jako
v případě organofosfátových chemických bojových látek.
METODIKA A VÝSLEDKY
Chemické látky
Sarin: (RS)-O-isopropylmethylfluorofosfát, GB látka, je irreverzibilní kompetitivní inhibitor
acetylcholinesterázy. Organofosfát sarin se kovalentně váže na -OH skupinu postranního
řetězce serinu v aktivním místě acetylcholinesterasy (3). Inhibice tohoto enzymu má za
následek sníženou schopnost těla hydrolyzovat acetylcholin v synaptické štěrbině zpět na
cholin a acetát (viz Syntéza acetylcholinu). Tudíž dlouhá expozice acetylcholinu na receptory
cholinergních synapsí brání opětné repolarizaci neuronu, a tím dalšímu vedení vzruchu.
Cholinergní receptory jsou dvojího druhu: nikotinové vyskytující se v nervosvalových
ploténkách a sympatických gangliích a muskarinové nacházející se v mozkových buňkách a
periferním nervstvu (4).
Tabun: O-ethyldimethylamidokyanofosfát, GA látka, stejně jako ostatní organofosfáty
ovlivňuje cholinergní přenos nervového vzruchu, který je na synapsi zprostředkován
neuromediátorem acetylcholinem. Základním mechanismem účinku je zásah do
cholinergního nervového systému cestou ireverzibilní inhibice acetylcholinesterázy - enzymu,
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který cholinergní neuromediátor rozkládá v centrálním i periferním cholinergním nervovém
systému. Ireverzibilní inhibice acetylcholinesterázy vede k narušení cholinergního přenosu
nervového vzruchu patologickým nahromaděním acetylcholin na receptorech s následným
dlouhodobým nadměrným drážděním cholinergních receptorů. Toxicita tabunu je při
vdechování asi poloviční oproti sarinu, ve velmi malých koncentracích však tabun dráždí oči
více než sarin. Tabun se navíc pomalu rozpadá, proto opakované expozice mohou vést k
akumulaci v těle (5).
Reaktivátory AChE: obidoxim (1,3-bis(4-hydroxyiminomethylpyridinium)-2-oxa-propan
dibromid), oxim HI-6 (1-(2-hydroxyiminomethylpyridinium)-3-(4-karbamoylpyridinium)-2oxa-propan dichlorid), a pralidoxim (2-hydroxyiminomethyl-1-methylpyridinium bromid)
Humanin: H2N-Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-LeuPro-Val-Lys-Arg-Arg-Ala-OH a jeho analogy mají schopnost ochrany mozkových buněk před
atakem AD genů a amyloid-beta-peptidů in vitro (6).
Zvířata
K experimentální práci byli jako pokusná zvířata vybráni samci laboratorních potkanů
kmene Wistar (Rattus norvegicus albino Wistar) získaných z chovného zařízení v
Konárovicích. Zvířata byla držena v klimatizované místnosti (teplota vzduchu 20-24ºC;
vlhkost vzduchu 50% ± 10) s řízeným světelným režimem 12/12 hodin a krmena standardní
peletovou potravou a vodou ad libitum. Vykonání experimentů a manipulace se zvířaty byla
provedena pod dohledem odborné rezortní komise v souladu s vyhláškou 207/2004 Sb. o
ochraně, chovu a využití pokusných zvířat. Personál manipulující se zvířaty byl předem
proškolen a obdržel osvědčení o způsobilosti pracovat s laboratorními zvířaty podle § 17
odst. 1 zákona.
T bludiště
Sestávající z několika segmentů o rozměru 12 x 10-20 x 11 cm. Nejkratší průchozí trasa je
délky 185 cm, v cílovém kompartmentu umístěno několik pelet jako pozitivní motivace
zvířat. Zaznamenán byl čas potřebný k překonání bludiště a počet chyb.
Morrisovo vodní bludiště
180 cm kruhový bazén pomyslně rozdělen do čtyř kompartmentů, naplněn vodou do výšky
hladiny 25 cm. Cílová platforma je 2 cm pod úrovní hladiny a pro testované zvíře vlivem
zrcadlového efektu hladiny neviditelná. Její pozice se mění dle schématu daného pokusu. Na
vnitřních stěnách bludiště jsou umístěné orientační znaky k navigaci zvířete.
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Y bludiště
Jednoduché bludiště ve tvaru Y se zabudovaným elektrickým aparátem pro averzivní
motivaci zvířete. Pokusné zvíře je vedeno k průchodu do toho ramene, nad kterým svítí
dioda na principu základního diskriminačního učení.
VÝSLEDKY: KONKRÉTNÍ STUDIE
ZÁVĚR
V jednotlivých studiích byly pro testování kognitivních procesů použity jednoduché Y bludiště
s averzivní motivací, komplexní T – bludiště s pozitivní motivací a Morrisovo vodní bludiště.
Pro svou kognitivně motorickou dispozici byl k pokusům použit jako animální model
laboratorní potkan kmene albino Wistar.
Byly provedeny série in vivo behaviorálních testů na potkanech pro hodnocení
neuroprotektivního účinku nových analogů humaninu – potencionálně léčivých látek
v terapii Alzheimerovy choroby. První série testů byla provedena metodou komplexního Tbludiště (testování schopnosti vybavit si dříve osvojenou kognitivní mapu) a otestovány byly
analogy: des-Leu-PAGA ve vyšší koncentraci (0,6 μmol.kg-1, i.p.), HL 607 (0,2 μmol.kg-1, i.p.)
a HL 608 (0,2 μmol.kg-1, i.p.). Dvě ze zkoumaných látek, a to HL 607 a HL 608, způsobily
výrazné zlepšení prostorové orientace potkanů, která byla poškozena podáním 3quinuclidinyl benzilátu (2 mg.kg-1, i.p), což prokazuje jejich neuroprotektivní účinek.
Podáním vyšší koncentrace látky des-Leu-PAGA, která byla pozitivně testována v roce 2003,
nebylo dosaženo většího účinku. V druhé polovině roku 2004 se podařilo zajistit nový přístroj
pro testování prostorové paměti a orientace laboratorních hlodavců – vodní Morrisovo
bludiště. Testování pomocí této metody umožňuje ještě objektivnější, přesnější a validnější
vyhodnocení výkonů testovaných zvířat, než stávající T- bludiště. Testována byla schopnost
osvojit si novou kognitivní mapu u naivních zvířat. V této druhé sérii behaviorálních testů
byly testovány látky HL 598 a HL 608. Výsledky testů prokázaly, že ani jedna z testovaných
látek negativně neovlivňuje motorickou výkonnost zvířat (rychlost plavání byla stejná u
ovlivněných jako kontrolních zvířat). V tomto testu se neprojevil neuroprotektivní účinek
látky HL 598 ani HL 608. Vzhledem k první sérii testu v tomto roce z našich výsledků vyplývá,
že látka HL 608 má protektivní účinek v případě následného udržení si dříve osvojené
kognitivní mapy, ale neúčinkuje v této koncentraci při osvojování si nové kognitivní mapy u
naivního zvířete.
V rámci vojenského výzkumu byly provedeny studie zaměřené na testování vlivu různých
reaktivátorů acetylcholinesterázy (obidoxim, trimedoxim, HI-6) při injekčním nebo
inhalačním podání subletálních dávek vojensky významných látek (tabun, sarin).
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Historicky popsané základní procesy učení živočichů jsou dodnes na různých úrovních
využívány v moderním preklinickém farmakologickém a medicínském výzkumu a vývoji
nových léčiv. Technický vzestup používaných přístrojů umožňuje přesnější rozlišení účinků
látek na základě sledovaných a zaznamenávaných reakcí a výkonů zvířat. Rovněž se s
postupem času a etickému významu více přistupuje ke komplexnějším a méně invazivním
metodikám, jednoduché přístroje na principu averzivní motivace (např. elektrické impulzy)
ustupují technicky náročnějším zařízením, která se svým provedením více blíží přirozeným
podmínkám, ve kterých daná laboratorní zvířata žijí.
REFERENCE
1. HYNIE S. Speciální farmakologie. Díl III - Látky ovlivňující CNS. 2. vyd. Praha : Karolinum
2000, 299 s.
2. BROWN, MA. KELLEY, AB. Review of health consequences from high-, intermediate- and
low-level exposureto organophosphorus nerve agents. J Appl Toxicol 1998; 18: 393-408.
3. MURRAY, R., GRANNER, D., MAYES, P., RODWELL, V. Harperova biochemie. 4. vyd.
Jinočany: Nakladatelství H+H 2002. 784 s.
4. LEDVINA, M., STOKLASOVÁ, A., CERMAN, J. Biochemie pro studující medicíny. 1. vyd.
Praha: Karolinum 2004, 473 s.
5. PATOČKA, J., a kol. Vojenská toxikologie, 1. vyd. Praha: Grada Publishing, 2004, 180 s.
6. HASHIMOTO Y. et al. PNAS 2001; 98: 6336-6341.
SEZNAM VYBRANÝCH AUTORSKÝCH A
SPOLUAUTORSKÝCH PUBLIKACÍ
KREJČOVÁ, G., et al. Effect of cyclosporine A on the activity of acetylcholinesterase in
selected parts of the rat brain. Homeostasis in Health and Diseases, 2001, vol. 41, no. 3-4, p.
162-163.
KREJČOVÁ, G. The effect of ovulatory cycle in African elephant on their behaviour. Elephant
Journal of EEKMA, 2002, vol. 5, no. 4, p. 3-6.
KREJČOVÁ, G., et al. Low-level sarin-induced alteration of memory and its treatment in rats.
Homeostasis in Health and Diseases, 2003, vol. 42, no. 1-2, p. 83-85.
KREJČOVÁ, G., et al. Comparison the efficacy of acetylcholinesterase reversible inhibitors and
acetylcholinesterase reactivator as the pretreatment for organophosphate intoxication.
7
Proceedings of the 8th International symposium on protection against chemical and
biological warfare agents. 2004, vol. 8. p. 1-6.
KREJČOVÁ, G., et al. Des-leu-Paga analogues-synthesis and effect on the experimentally
induced impairment of spatial memory in multiple T-maze test in rats. Journal of peptide
science, 2004, vol. 10, Suppl. 1, p. 171.
KREJČOVÁ, G., et al. In vivo comparison of centrally and peripherally acting drugs efficacy as
the functional antidotes for the treatment of experimentally organophosphate-induced
intoxication. Proceedings of the 7th International ISSX meeting, 2004, vol. 36, Suppl. 1, p.
330.
KREJČOVÁ, G., et al. Neuroprotektivní účinek analogů humaninu v případě experimentálně
navozeného poškození prostorové paměti potkanů. Psychiatrie, 2004, roč. 8, Suppl. 1, s. 43.
KREJČOVÁ, G., et al. Účinek atropinu a oximu HI-6 na výkony potkanů ovlivněných nízkou
dávkou sarinu v T bludišti. Psychiatrie, 2003, roč. 7, č. 1, s. 30.
KREJČOVÁ, G., HERINK, J. Současné poznatky o CYKLOSPORINU A. Vojenské zdravotnické
listy, 2002, roč. 71, č. 3, s. 115-120.
KREJČOVÁ, G., HERINK, J., BAJGAR, J. Efekt cyklosporinu na inhibiční aktivitu 7methoxytakrinu. Psychiatrie, 2002, roč. 6, supp. 1, s. 27.
KREJČOVÁ, G., KASSA, J. Neuroprotective efficacy of pharmacological pretreatment and
antidotal treatment in tabun-poisoned rats Toxicology, 2003, vol. 185, p. 129-139.
KREJČOVÁ, G., KASSA, J. Neuroprotektivní účinnost farmakologické profylaxe a antidotní
léčby u experimentální intoxikace tabunem. Sborník abstrakt z 8. Česko-slovenské
mezioborové toxikologické konference, 2003, roč. 8, s. 26.
KREJČOVÁ, G., KASSA, J. Ovlivnění prostorové paměti potkanů nízkými dávkami sarinu.
Sborník abstrakt 30. české a slovenské konference, 2003, roč. 30. s. 47.
KREJČOVÁ, G., KASSA, J. Acute experimental tabun-induced intoxication and its therapy in
rats. Central European Journal of Public Health. 2004, vol. 12, Suppl. 1, p. 48-52.
KREJČOVÁ, G., KASSA, J. Anticholinergic drugs-functional antidotes for the treatment of
tabun intoxication. Acta Medica (Hradec Králové), 2004, vol. 47, no. 1, p. 13-18.
KREJČOVÁ, G., KASSA, J. Comparison of anticholinergic drugs as the functional antidotes for
the treatment of tabun-induced intoxication. Proceedings of the Fifth International Chemical
and Biological Medical Treatment Symposium, 2004, vol. 5, p. 172-178.
KREJČOVÁ, G., KASSA, J. SAMNALIEV, I.M. Využití biperidenu při léčbě experimentální akutní
intoxikace somanem. Československá fyziologie, 2003, roč. 52, č. 4, s. A13.
8
KREJČOVÁ, G., KASSA, J., VACHEK, J. Effect of atropine and the oxime HI-6 on low-level sarininduced alteration of performance of rats in a T-maze. Acta Medica (Hradec Králové), 2002,
vol. 45, no. 3, p. 107-110.
KREJČOVÁ, G., KUČA, K,. ŠEVELOVÁ, L. Cyclosarin - current position of an organophosphate
nerve agent. Defence Science Journal, 2005, vol. 55, no. 2, p. 1-12.
KREJČOVÁ, G., PATOČKA, J., SLANINOVÁ, J. Effect of humanin analogues on experimentally
induced impairment of spatial memory in rats. Journal of Peptide Science, 2004, vol. 10, no.
10, p. 636-639.
KREJČOVÁ, G., ŠEVELOVÁ, L. Současné poznatky o galantaminu, reverzibilním inhibitoru
acetylcholinesterázy. Vojenské zdravotnické listy, 2003, roč. 72, č. 1, s. 37-44.
KREJČOVÁ, G., ŠEVELOVÁ, L., KUČA, K. Cyklosarin v kontextu dnešní doby. Zpravodaj
vojenské farmacie, 2004, č. 2, s. 65-11.
KREJČOVÁ, G., ŠEVELOVÁ, L., KUČA, K. The oxime HS-6: A potential point for therapy of
cyclosarin-induced intoxication. Proceedings of the Fifth International Chemical and
Biological Medical Treatment Symposium, 2004, vol. 5, p. 179-181.
KUNEŠOVÁ, G., et al. The evaluation of tabun-induced cognitive impairment in rats and its
antidotal treatment using the water maze test. Homeostasis in Health and Diseases, 2004,
vol. 43, no. 2, p. 82-85.
KUNEŠOVÁ, G., et al. Efficacy of new des-leu-PAGA analogues on the experimentally induced
impairment of spatial memory in multiple t-maze test in rats. Psychiatrie, 2005, roč. 9, supp.
1, s. 42.
KUNEŠOVÁ, G. Et al. The multiple T-maze in vivo testing of the neuroprotective effect of
humanin analogues. Peptides, 2008, vol. 29, no. 11, p. 1982-7.
HERINK, J., KREJČOVÁ, G., BAJGAR, J., et al. Cyclosporine A inhibits acetylcholinesterase
activity in selected parts of the rat brain. Neuroscience Letters, 2003, vol. 339, p. 251-253.
HERINK, J., KREJČOVÁ, G., BAJGAR, J. Antiacetylcholinesterase activity of cyclosporine - a
comparison of single and repeated administration and effect of 7-methoxytacrine. Acta
Medica (Hradec Králové), 2002, vol. 45, no. 4, p. 145-147.
KASSA, J., KREJČOVÁ, G. Neuroprotective effects of currently used antidotes in tabunpoisoned rats. Pharmacology & Toxicology, 2003, vol. 92, p. 258-264.
KASSA, J., KREJČOVÁ, G. The influence of acetylcholinesterase reactivator selection on the
elimination of tabun-induced signs of neurotoxicity in rats. Homeostasis in Health and
Diseases, 2003, vol. 42, no. 1-2, p. 79-82.
9
KASSA, J., KREJČOVÁ, G. Neuroprotective effects of currently used antidotes in tabunpoisoned rats. Proceedings of the Fifth International Chemical and Biological Medical
Treatment Symposium, 2004, p. 141-145.
KASSA, J, KREJČOVÁ, G., SAMNALIEV, I. A comparison of the neuroprotective efficacy of
pharmacological pretreatment and antidotal treatment in soman-poisoned rats. Acta Medica
(Hradec Králové), 2003, vol. 46, no. 3, p. 101-107.
KASSA, J., KREJČOVÁ, G., VACHEK, J. The influence of low-level sarin inhalation exposure on
the spatial memory of rats. Homeostasis in Health and Diseases , 2001, vol. 41, no. 3-4, p.
157-159.
KASSA, J., KREJČOVÁ, G., VACHEK, J. The impairment of spatial memory following low-level
sarin inhalation exposure and antidotal treatment in rats. Acta Medica (Hradec Králové),
2002, vol. 45, no. 4, p. 149-153.
KASSA, J., KUNEŠOVÁ, G. Vliv složení antidotní terapie na eliminaci tabunem vyvolaných
poruch kognitivních funkcí. Psychiatrie, 2005, roč. 9, supp. 1, s. 41.
ŠEVELOVÁ, L., KUČA, K., KREJČOVÁ-KUNEŠOVÁ, G. Antidotal treatment of GF-agent
intoxikation in mice with bispyridinium oximes. Toxicology, 2005, vol. 207, p. 1-6.
Evangelou, A., Zikos, C., Benaki, D., Pelecanou, M., Bouziotis, P., Papadopoulos, M.,
Borovickova, L., Vesela, I., Elbert, T., Kunešová, G., Pirmettis, I., Paravatou-Petsotas, M.,
Slaninová, J., Livaniou, E. Peptides, 2009, vol. 12, no. 30, p. 2409-2417.
PATOČKA, J., SLANINOVÁ, J., KUNEŠOVÁ, G. Neuroprotective peptides as drug candidates
against Alzheimer's diasease. Journal of Applied biomedicine, 2010, vol. 3, no. 2, p. 67-73.
10
Journal of Peptide Science
J. Peptide Sci. 10: 636–639 (2004)
Published online 7 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.569
Effect of Humanin Analogues on Experimentally Induced
Impairment of Spatial Memory in Rats
GABRIELA KREJCOVA,a *JIRI PATOCKAa and JIRINA SLANINOVAb
a
Department of Toxicology, Purkyně Military Medical Academy, Třebešská 1575, 500 01 Hradec Králové,
Czech Republic
b
Institute of Organic Chemistry and Biochemistry AVČR, Flemingovo sq. 2, 16610 Prague, Czech Republic
Received 4 December 2003
Accepted 19 January 2004
Abstract: Humanin and its analogues have been shown to protect cells against death induced by various
Alzheimer’s disease genes and amyloid-β-peptides in vitro; the analogue [Gly14 ]-humanin has also been
shown to be potent in reversing learning and memory impairment induced by scopolamine in mice in vivo.
It is important to validate these results by using other behavioral methods. In this study, the effect of
[Gly14 ]-humanin and des-Leu-PAGA, another analogue (0.2 µmol kg−1 , i.p.) on the 3-quinuclidinyl benzilateinduced (2 mg kg−1 , i.p.) impairment of spatial memory in the multiple T-maze in rats has been evaluated.
Both peptides reversed the impairment of spatial memory. These results indicate the potential of humanin
analogues in modulation of the cholinergic system. Copyright  2004 European Peptide Society and John
Wiley & Sons, Ltd.
Keywords: humanin; [Gly14 ]-humanin; humanin analogue; memory; T-maze test; Alzheimer’s disease;
3-quinuclidinyl benzilate
INTRODUCTION
Humanin (Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-LeuLeu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-LysArg-Arg-Ala; MW = 2686.3; C119 H203 N34 O32 S2 ) and
its analogues have been shown to protect neuronal
cells against death induced by various Alzheimer’s
disease (AD) genes and amyloid-β-peptides in vitro
[1–5]. However, testing their in vivo effect is desirable. Direct suppression of neuronal cell death
would be promising for the development of antiAD therapeutics [2]. For the evaluation of the effect
of compounds on memory functions, i.e. memory
formation, consolidation, impairment, on long-term
and short-term memory etc, there are several behavioral tests [6]. Only one study of the effect of the
* Correspondence to: Gabriela Krejcova, Department of Toxicology,
Purkyně Military Medical Academy, Třebešská 1575, 500 01 Hradec
Králové, Czech Republic; e-mail: [email protected]
Contract/grant sponsor: Grant Agency of the Czech Republic;
Contract/grant number: 305/03/1100.
Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd.
humanin analogue [Gly14 ]-humanin in vivo in the
Y-maze test after i.c.v. administration in mice has
appeared [7]: it showed the analogue to be potent
in reversing the impairment of spontaneous alternation behaviour induced by scopolamine in the
Y-maze, an index of short-term memory in mice.
It is important to validate these results by using
other behavioral methods. In this study, the effect
was evaluated of [Gly14 ]-humanin and the humanin
analogue des-Leu-PAGA on 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation
and memory in the multiple T-maze in rats. QNB
is an anticholinergic drug which affects both the
peripheral and central nervous system. It is a potent
competitive inhibitor of acetylcholine at postganglionic muscarinic synaptic sites, although in high
doses it can also affect nicotinic sites [8]. Pharmacologically, it acts on the central and peripheral
nervous systems like scopolamine. It is able to cross
the blood–brain barrier. This drug is commonly
HUMANIN ANALOGUES
used for experimental modeling of memory deficits
in animals [9–11].
MATERIALS AND METHODS
Peptides
[Gly14 ]-Humanin from Clonestar Biotech (Brno,
Czech Republic) was re-purified on semipreparative
HPLC and gave a single peak on analytical
HPLC; MS confirmed the molecular weight. DesLeu-PAGA (Pro-Ala-Gly-Ala-Ser-Cys-Leu-Leu-LeuThr-D-Ser-Glu-Ile-Asp-Leu-Pro) was obtained from
PolyPeptide Laboratories and showed also a single
peak on analytical HPLC; MS confirmed the
molecular weight. PAGA is a shorter active core
(3–19) analogue of humanin (the name comes
from the first four amino acids in the sequence,
Pro-Ala-Gly-Ala) [12]. Both peptides were dissolved
in physiological saline to give 0.2 mM solutions.
QNB was synthesized at Purkyně Military Medical
Academy Laboratory.
Animals
Male albino Wistar rats weighing 180–200g were
obtained from VÚFB Konárovice (Czech Republic).
They were kept in an air-conditioned room and
maintained on a 20 h food deprivation schedule
with food available only in the maze and for 4 h
after the daily trial. Tap water was available ad
libitum. Handling of the experimental animals was
done under supervision of the Ethics Committee
of the Medical Faculty of Charles University and
the Purkyně Military Medical Academy in Hradec
Králové (Czech Republic).
Multiple T-maze test
The T-maze [13,14] consisted of five segments 12 cm
wide, 20 cm long and 11 cm high. One of the
arms (the goal compartment) contained a reward
(several food pellets), the length of the correct
track from start to the goal compartment was
185 cm. The rats were trained to pass the maze
in less than 3 min without entering the wrong
arm once a day for 20 days. Only rats that for
four successive days had not made any mistake
were used in the experiment. The rats were divided
into ten groups of seven animals. QNB (2 mg kg−1 )
was injected 15 min after the peptide injection
(0.2 µmol/kg). All substances were administered
Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd.
637
intraperitoneally. Cognitive functions were tested
before drug application and then 15 min, 24 h and
7 days following the QNB injection. Controls were
tested at the same time intervals after physiological
saline or peptide administration alone. The number
of entries into the wrong arms, and passage times
through the maze were recorded. Statistical analysis
was performed on a PC with Statistica software
‘98 Edition. Analysis of variance (ANOVA) and
the Scheffé method of contrasts were used for
the determination of significant differences between
the control and experimental groups [15]. The
differences were considered significant when p <
0.05.
RESULTS
Our study confirmed that the performance in
the T-maze of rats injected i.p. with QNB alone
was changed in comparison to that before the
injection or to that of control animals (rats injected
with physiological saline). The rats showed a
high number of entries into wrong arms of the
maze, and prolonged passage times through the
maze. Aggravation of the T-maze performance was
observed 15 min and 24 h after drug application
(Figure 1). Both peptides significantly attenuated
the impairment of the performance, i.e. the spatial
memory and orientation when injected 15 min
before QNB: there was a reduction of passage time
through the maze (Figure 1) in comparison with QNB
alone. Although 15 min following QNB injection,
the number of wrong entries by the group of
[Gly14 ]humanin- and QNB-treated rats was similar
to that of the group which received QNB alone,
the peptide-treated animals were able speedily to
remedy their mistakes. 24 h after treatment, there
was a statistically significant reduction in the
number of wrong entries (Table 1) and passage times
(Figure 1) if the rats were pretreated with either
peptide.
DISCUSSION
This study examined the effect of [Gly14 ]humanin
and des-Leu-PAGA-humanin on spatial memory and
orientation impairment induced by QNB. This drug
is used for experimental induction of central anticholinergic syndrome [16] with a negative impact
on memory and learning [17,18]. Cholinergic neuronal systems play an important role in the cognitive
J. Peptide Sci. 10: 636–639 (2004)
KREJCOVA, PATOCKA AND SLANINOVA
638
##
200
control I (physiological solution)
QNB alone
control II (peptide only)
des-Leu-PAGA + QNB
[Gly14]-humanin + QNB
TIME (s)
150
100
#
#
*
50
**
**
*
*
*
0
before drug application
15 min
24 h
7d
TIME of TESTING
Figure 1 Performance of rats in the T-maze — passage times of the variously treated groups of rats before and 15 min,
24 h and 7 days after drug injection (statistical significance: # and ## vs control I; p < 0.05 and p < 0.01, respectively; ∗
and ∗∗ vs QNB alone, p < 0.05 and p < 0.01, respectively).
Table 1 Performance of Rats in the T-maze — Number of Wrong Entries of Variously Treated Rats with
Statistical Differences (statistical significance a vs control I, p < 0.05; b vs QNB alone, p < 0.05)
Group of rats
Number of error entries
Before drug application
After drug application
15 min
Control I (n = 7)
QNB alone (n = 7)
Control II (n = 7)
des-Leu-PAGA + QNB (n = 7)
[Gly14 ]-humanin + QNB (n = 7)
24 h
7d
Ø
±s
Ø
±s
Ø
±s
Ø
±s
0
0
0
0
0
0
0
0
0
0
0
2.57a
0
1.29a
3.00a
0
2.76
0
1.61
2.77
0
0.57a
0b
0b
0b
0
0.13
0
0
0
0.29
0.14
0.29
0.43
0.14
0.49
0.38
0.49
0.54
0.38
deficits associated with AD and other neurodegenerative diseases [19]. The study of spatial recognition
and memory is a useful method to evaluate potentially antiamnesic drugs [13]. In our study, all drugs
were injected i.p., and were found to be active. The
fact that the peptides were active after i.p. administration is very significant for drug development.
Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd.
No evidence that humanin analogues cross the
blood–brain barrier has so far been published, and
the finding of a central effect of humanin analogues
following administration in our experiment is therefore fundamental. Peptides are generally rapidly
degraded by gastrointestinal enzymes, and thus
injection is the preferred route of administration.
J. Peptide Sci. 10: 636–639 (2004)
HUMANIN ANALOGUES
This is valid also for humanin [20]. Both peptides
used had a significant antiamnesic effect — they
reversed memory impairment induced by QNB in
rats. Our results confirm the antiamnesic potential
of [Gly14 ]-humanin and demonstrate the neuroprotective effect of des-Leu-PAGA. This humanin analogue has a shorter chain length than humanin (16
versus 24 residues) and lacks one leucine in the middle sequence of the molecule (it has three successive
leucines instead of the four present in humanin).
This feature simplifies the synthesis (unpublished
observation). These results indicate the potential
of humanin analogues for the modulation of the
cholinergic system and shows that they are able to
improve both short-term and long-term memory in
rodents.
Acknowledgement
The work was supported by the Grant Agency of
Czech Republic No. 305/03/1100.
REFERENCES
1. Hashimoto Y, Niikura T, Tgima H, Yasukawa T,
Sudo H, Ito Y, Kita Y, Kawasumi M, Kouyama K,
Doyu M,
Sobue G,
Koide T,
Tsuji S,
Lang J,
Kurokawa K, Nishimoto I. A rescue factor abolishing
neuronal cell death by a wide spectrum of familial
Alzheimer’s disease genes and aβ. PNAS 2001; 98:
6336–6341.
2. Niikura T, Hashimoto Y, Tajima H, Nishimoto I. Death
and survival of neuronal cells exposed to Alzheimer’s
insults. J.Neurosci. Res. 2002; 70: 380–391.
3. Tajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y,
Terashita K, Yamazaki K, Koto A, Aiso S, Nishimoto I.
Evidence for in vivo production of Humanin peptide,
a neuroprotective factor against Alzheimer’s diseaserelated insults. Neurosci. Lett. 2002; 324: 227–231.
4. Kawasumi M, Hashimoto Y, Chiba T, Kanekura K,
Yamagishi Y,
Ishizaka M,
Tajima H,
Niikura T,
Nishimoto I. Molecular mechanisms for neuronal cell
death by Alzheimer’s amyloid precursor proteinrelevant insults. Neurosignals 2002; 11: 236–250.
5. Kariya S, Takahashi N, Ooba N, Kawahara M,
Nakayama H, Ueno S. Humanin inhibits cell death
of serum-deprived PC12h cells. Neuroreport 2002; 13:
903–907.
Copyright  2004 European Peptide Society and John Wiley & Sons, Ltd.
639
6. Bureš J, Burešová O, Křivánek J (eds). Brain and
Behavior: Paradigms for Research in Neural
Mechanisms, 1st edn. Academia: Praha, 1988;
151–236.
7. Mamiya T, Ukai M. [Gly14 ]-Humanin improved the
learning and memory impairment induced by
scopolamin in vivo. Br. J. Pharmacol. 2001; 134:
1597–1599.
8. http://www. mobrien.com/twr/bz.htm
9. Mezey Sz, Székely AD, Bourne RC, Kabai P, Csillag A.
Changes in binding to muscarinic and nicotinic
cholinergic receptors in the chick telencephalon,
following passive avoidance learning. Neurosci. Lett.
1999; 270: 75–78.
10. Amenta F, Cavalloti C, Franch F, Ricci A. Muscarinic
cholinergic receptors in the hippocampus of the aged
rat: effects of long-term hydergine administration.
Arch. Int. Pharmacodyn. Ther. 1989; 297: 225–234.
11. Benishin CG, Lee R, Wang LCH, Liu HJ: Effects of
ginsenoside-RB1 on central cholinergic metabolism.
Pharmacology 1991; 42: 223–229.
12. Yamagishi Y, Hashimoto Y, Niikura T, Nishimoto I.
Identification of essential amino acids in Humanin,
a neuroprotective factor against Alzheimer’s diseaserelevant insults. Peptides 2003; 24: 585–595.
13. Bureš J, Burešová O, Huston J (eds). Techniques and
Basic Experiments for the Study of Brain and Behavior,
1st edn. Elsevier: Amsterdam, 1979; 147–150.
14. Krejcova G, Kassa J, Vachek J. Effects of atropine and
the oxime HI-6 on low-level sarin-induced alteration
of performance of rats in a T-maze. Acta Med. (Hradec
Králové) 2002; 45: 107–110.
15. Afifi AA, Azen SP (eds). Statistical Analysis and
Computer Oriented Approach, 2nd edn. Academic
Press: New York, 1979; 442–445.
16. Schneck HJ, Rupreht J, Waldvogel HH, Hundelshausen BV. The central anticholinergic syndrome.
J. Anaesth. Clin. Pharmac. 1995; 11: 267–273.
17. Abood LG. Some new approaches to studying the mode
of action of central nervous system poisons. J. Med.
Pharmac. Chem. 1961; 4: 469–481.
18. Albanus L. Studies on central and peripheral effects of
anticholinergic drugs. FOA Reports 1970; 4: 1–17.
19. Bartus AT, Dean RL, Beer B, Lippa AS. The cholinergic
hypothesis of geriatric memory dysfunction. Science
1982; 217: 408–417.
20. Hashimoto Y, Ito T, Niikura T, Shao Z, Hata M,
Oyama F, Nishimoto I. Mechanisms of neuroprotection
by a novel rescue factor Humanin from Swedish
mutant amyloid precursor protein. Biochem. Biophys.
Res. Commun. 2001; 283: 460–468.
J. Peptide Sci. 10: 636–639 (2004)
peptides 29 (2008) 1982–1987
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
The multiple T-maze in vivo testing of the neuroprotective
effect of humanin analogues
Gabriela Kunešová a, Jan Hlaváček b, Jiřı́ Patočka c, Alexandra Evangelou d,
Christos Zikos d,e, Dimitra Benaki f, Maria Paravatou-Petsotas d,
Maria Pelecanou f, Evangelia Livaniou d, Jirina Slaninova b,*
a
Department of Toxicology, University of Defense, Třebešská 1575, 500 01 Hradec Králové, Czech Republic
Institute of Organic Chemistry and Biochemistry AVČR, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
c
Department of Radiology and Toxicology, Faculty of Health and Social Studies, University of South Bohemia,
České Budějovice, Czech Republic
d
Institute of Radioisotopes and Radiodiagnostic Products, NCSR ‘‘Demokritos’’, 153 10 Athens, Greece
e
Biomedica Life Sciences, Athens, Greece
f
Institute of Biology, NCSR ‘‘Demokritos’’, 153 10 Athens, Greece
b
article info
abstract
Article history:
Humanin (HN) and its analogues have been shown to protect cells against death induced by
Received 28 April 2008
various Alzheimer’s disease (AD) genes and amyloid-b-peptides in vitro; the analogues [Gly14]-
Received in revised form
HN and colivelin have also been shown to be potent in reversing learning and memory
25 June 2008
impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo
Accepted 25 June 2008
using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of
Published on line 5 July 2008
the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the
multiple T-maze test in rats. The following peptides have been studied: HN analogues
Keywords:
truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in
Humanin-like peptides
the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its
Colivelin
analogues having three or five leucines instead of four, and finally the recently described hybrid
Rat
peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA
Memory
attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues.
Alzheimer’s disease
While the truncated analogues and most of the tert-Leu containing analogues were devoid of
Quinuclidinyl benzilate
activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial
memory irrespective of the number of Leu present in their sequence. The highest activity was
shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN
analogues in the modulation of the cholinergic system, which plays an important role in the
cognitive deficits associated with AD and other neurodegenerative diseases.
# 2008 Elsevier Inc. All rights reserved.
1.
Introduction
Alzheimer’s disease (AD) is the most common cause of
dementia [6]. Central changes in AD include, in addition to
the formation of amyloid plaques and neurofibrillary tangles,
huge and progressive loss of neurons [24]. The mechanism of
neuronal death and especially its relationship with accumulation and precipitation of pathogenic proteins is still a mystery;
* Corresponding author at: Biologically Active Peptides, Institute of Organic Chemistry and Biochemistry AVCR, Flemingovo sq. 2, 166 10
Prague 6, Czech Republic. Tel.: +420 220 183 317; fax: +420 220 183 578.
E-mail address: [email protected] (J. Slaninova).
0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2008.06.019
peptides 29 (2008) 1982–1987
therefore, for the time being one of the most promising
therapeutic approaches against AD focuses on neuroprotection [23].
Several sorts of substances have been tested for neuroprotective activity, such as inhibitors of acetyl cholinesterase,
cholesterol synthesis reducing substances, inhibitors of
proteolytic enzymes or anti-inflammatory agents. Neurosteroids, alkaloids, lipids, peptides and special chimeras constructed by bridging peptides actively entering the brain and
peptides acting as neuroprotective agents are among the
substances investigated for neuroprotection [11,26].
Seven years ago a new peptide called humanin (HN) has
been discovered by a Japanese group through functional
expression screening in human brain [13,14]. HN (Met-Ala-ProArg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-AspLeu-Pro-Val-Lys-Arg-Arg-Ala) and its analogues have been
shown to protect neuronal cells against death induced by
various AD genes and amyloid-b-peptides in vitro [13–
16,24,29,30].
The structure–activity study on the HN family peptides
resulted in an ‘‘active core peptide’’, the heptadecapeptide
PAGASRLLLLTGEIDLP (RG-PAGA), with in vitro activity 1000
times higher than that of humanin [25]. These results point to
the fact that clever amino acid modifications of the peptide
chain might lead to a substance usable as drug against this
very serious and widely encountered disease of the developed
world. In addition to the in vitro assays, several behavioral tests
have been used for the in vivo evaluation of the effect of HN
peptides on memory functions, e.g. memory formation,
consolidation, impairment of long-term, short-term memory,
etc. [7]. The first study described the in vivo effect of the HN
analogue [Gly14]-HN in the Y-maze test (index of short-term
1983
memory) after i.c.v. administration in mice [20] and showed
the analogue to be potent in reversing the impairment of
spontaneous alternation behavior induced by scopolamine.
Our group has subsequently shown [19] that the NH analogues
[Gly14]-HN and des-Leu-PAGA (PAGASCLLLTsEIDLP) are also
active after i.p. administration on 3-quinuclidinyl benzilate
(QNB)-induced impairment of spatial orientation and memory
in the multiple T-maze in rats. Recently, our protocol of drug
administration was applied to mice using Y-maze for in vivo
evaluation of HN analogues [8]. Both T-maze and Y-maze tests
evaluate spatial orientation and memory; however the Y-test
is based on spontaneous behavior and T-test on motivated
learned tasks.
In this study, we have evaluated the effect of various
analogues of HN (peptides I–XIV, Scheme 1) on the QNBinduced impairment of spatial orientation and memory in rats
employing the T-maze test. First, we were interested in
investigating whether the number of the leucine residues in
the middle region of various N- or C-terminally truncated HN
analogues affects neuroprotective activity. We further investigated whether introduction of a tert-leucine (Tle, tert-butylglycine) [4] in place of one of the successive leucines in the
middle region of the sequence will affect the activity. Moreover, the effect of colivelin (SALLRSIPAPAGASRLLLLTGEIDLP),
i.e. a peptide having the activity-dependent neurotrophic
factor ADNF-9 attached to the N-terminus of the RG-PAGA
(active core of HN) [16] and its analogues with three or five
leucines in their middle region were evaluated in our test. As
mentioned above, the T-maze is a suitable tool for testing the
procedural knowledge and spatial orientation functions. QNB
is an anticholinergic drug that acts on both the peripheral and
central nervous system like scopolamine and is commonly
Scheme 1
1984
peptides 29 (2008) 1982–1987
used for experimental modeling of memory deficits in animals
[22]. It is a potent competitive inhibitor of acetylcholine at
postganglionic muscarinic synaptic sites, although in high
doses it can also affect nicotinic sites [17].
The HPLC fractions containing the products were pooled and
lyophilized. All peptides were characterized by analytical RPHPLC and capillary electrophoresis, and identified by ESI-MS.
2.2.
2.
Materials and methods
2.1.
Peptides
The peptides synthesized and tested are presented in Scheme
1.
Peptides I–VIII were synthesized on Fmoc-Pro-Tentagel-SPHB using the Fmoc strategy on an automatic synthesizer.
Coupling was performed using TCTU/DIPEA at excess (3 eq)
and cleavage (removal) from resin and deprotection was
performed using TFA/water/TIS/EDT (92.5:2.5:2.5:2.5) for 2 h.
The cleaved peptides were concentrated and precipitated by
ether and were further purified using HPLC (Cromasil C18
column; gradient solvents: A, 0.1% TFA/water and B, 0.1% TFA/
acetonitrile; UV detection, 220 nm). Peptides IX–XIV were also
synthesized with the Fmoc strategy as previously described
for HN [12] and were purified using semi-preparative RP-HPLC.
Animals
Male albino Wistar rats weighing 180–200 g were obtained
from VÚFB Konárovice (Czech Republic). They were kept in an
air-conditioned room and maintained on a 20-h food
deprivation schedule with food available only in the maze
and for 4 h after the daily trial. Tap water was available ad lib.
Handling of the experimental animals was done under the
supervision of the Ethics Committee of the Institute.
2.3.
T-maze test
The T-maze consisted of several segments 12 cm wide, 10–
20 cm long and 11 cm high. One of the arms (the goal
compartment) contained a reward (several food pellets), the
length of correct track from start to the goal compartment was
185 cm [19,18]. The rats were trained to pass the maze in less
than 3 s without entering the wrong arm once a day for 20
days. Only rats that had not made any mistake for four
Fig. 1 – Performance of rats in the T-maze. Passage times of the variously treated groups of rats before the experiment and
15 min, 24 h and 7 days after QNB injection (30 min after peptide administration). Statistical significance: (***) QNB vs.
control p < 0.005; (+++) peptide vs. QNB p < 0.005; (+) peptide vs. QNB p < 0.05). Peptides tested and doses: (a) [The10]PAGA
(IV) 0.2 mmol/kg, des-Leu-[Tle7]PAGA (VIII) 0.1 m/kg and des-Leu-[Tle7]PAGA (VIII) 1 mg/kg.(b) des-Leu-RG-PAGA (IX)
0.2 mmol/kg, RG-PAGA (X) 0.2 mmol/kg, RG-PAGA (X) 1 mmol/kg. (c) plus-Leu-PAGA-RG (XI) 1 mmol/kg, colivelin (XII) 0.2 mmol/
kg, and des Leu-colivelin (XIII) 0.2 mmol/kg. (d) plus-Leu-colivelin (XIV) 0.2 mmol/kg, and des-Leu-colivelin (XIII) 0.1 mmol/kg.
1985
peptides 29 (2008) 1982–1987
successive days were used in the experiment. The experimental animals were then divided into groups of eight.
The substance with negative impact to memory and
learning QNB (2 mg/kg) was injected 15 min after the peptide
injection (0.1–1.0 mmol/kg, i.e. 20–200 nmol/rat). All substances were administered intraperitoneally. Cognitive functions were tested before drug application and then 15 min,
24 h and 7 days following the QNB injection. Controls were
tested at the same time intervals after physiological saline or
peptide administration alone. The number of entries into the
wrong arms, and passage times through the maze were
recorded. Statistical analysis was performed on a PC with
Statistica software’98 Edition. Analysis of variance (ANOVA)
and the Scheffé method of contrasts were used for the
determination of significant differences between control and
experimental groups [1]. The differences were considered
significant when p < 0.05.
3.
Results
Our results are summarized in Fig. 1a–d and in Table 1. The
performance of rats injected i.p. with QNB alone in the T-maze
was significantly different in comparison to that before the
injection or to that of control animals (rats injected with
physiological saline or peptide only, as mentioned in the
procedure paragraph of Section 2). The rats showed prolonged
passage times through the maze and a high number of entries
into wrong arms of the maze. Aggravation of the T-maze
performance was observed 15 min and 24 h after QNB
application. The effect after 7 days was not statistically
significant. Two parameters were followed—passage time
through the maze and number of error entries (number of
entries into wrong arms of the maze). As far as the passage
time is concerned, positive effect was seen with the humanin
analogues des-Leu-[Tle7]PAGA (VIII), des-Leu-RG-PAGA (IX),
RG-PAGA (X), and plus-Leu-RG-PAGA (XI), colivelin (XII) and
des-Leu-colivelin (XIII). The positive effect of VIII, IX, X and XI
was comparable to the effect of des-Leu-PAGA described
previously [19]. The highest effect was seen with the colivelin
(XII) and des-Leu-colivelin (XIII) which showed statistically
significant improving effect both after 15 min and after 24 h
(Fig. 1c and d).
Substances I–IV were without any influence on the
aggravation effect of QNB. The effect of substances VI and
VII (not shown) and XIV (Fig. 1d) was not statistically
significant.
As far as the number of error entries is concerned (Table 1),
a tendency towards fewer entries into wrong arms could be
seen for some of the analogues, however the effect was not
always statistically significant. Statistically significant
improvement however on low level ( p < 0.05) showed compounds I, IX, XI, XII and XIII.
4.
Discussion
In this study, we examined the effect of peptides of the
humanin family on spatial memory and orientation impairment induced by QNB in rats. This drug is used for
experimental induction of central anticholinergic syndrome
with negative impact on memory and learning [28]. The pilot
studies focused on the effect of QNB to animal behavior (using,
e.g. Rota Rod and functional observational battery) showed
Table 1 – Performance of rats in the T-maze—number of wrong entries of the variously treated groups of rats before and
15 min, 24 h and 7 days after QNB (before, 30 min, 24 h and 7 days after peptide) injection
Compound
n (number of rats)
Number of error entries
Average
Control
QNB
I
II
III
IV
Ve
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
a
b
c
d
*
7/8
7/8
8
8
8
7
7
7
7
7
7
7
7
7
7
7
Before drug
30 min
24 h
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.57–7.17b
0.75*
1.75
3.38
2.50
1.29
1.29
1.86
1.43
1.29*
2.14
0.66*
0.71*
0.33*
3.71
0
0.29–1.33c
0*
0.13
0.13
0
0*
0.11
0.11
0.29
0*
0*
0*
0*
0*
0.28
0.25 for compounds I, II, III, V, VI and VII; 0 for compounds IX, X, XI, XII, and XIII.
2.57 for compounds I, II, III, V, VI and VII; 3.86 for compounds IX and X; 7.17 for compounds XI, XII, XIII and XIV.
0.29 for compounds IX and X; 0.57 for compounds I, II, III, V, VI and VII; 1.33 for compounds XI, XII, XIII and XIV.
0 for compounds IX, X, XI, XII, XIII and XIV; 0.14 for compounds I, II, III, V, VI and VII.
Means statistically significant effect vs. QNB group on the level p < 0.05.
7 days
0–0.25a
0–0.14d
0
0
0
0
0.43
0
0
0
0
0
0
0
0
0
1986
peptides 29 (2008) 1982–1987
that doses presented in our recent examination do not induce
statistical significant alterations in sensorial and/or motor
function. Cholinergic neuronal systems play an important role
in the cognitive deficits associated with AD and other
neurodegenerative diseases [2]. Loss of cholinergic markers
is often seen in patients with dementia, suggesting that
decreased cholinergic function could contribute to both the
cognitive deficits, and perhaps the neuronal degeneration
associated with dementia [5,27].
In our structure–activity study we first concentrated on the
middle lipophilic part of the sequence of humanin where a
group of four leucines is situated. We have synthesized a
series of analogues of the active core of humanin called PAGA
truncated at the C-end and having three, two or one leucine in
the middle part instead of four (analogues I, II, and III). Further
we synthesized analogues of the active humanin derivative
des-Leu-PAGA (V, already tested for its in vivo activity [19]) by
substituting individual leucines with the non-coded isosteric
amino acid tert-leucine (Tle). The presence of Tle is supposed
to reduce the flexibility of the peptide chain and thus affect its
conformation (analogues VI, VII, and VIII). This modification
may also result in higher resistance to enzymatic cleavage. In
addition, a longer form of humanin analogue being truncated
only at the N-terminus and having one leucine from the four in
the center replaced by Tle, was synthesized (analogue IV). Of
all these analogues, the only one that showed statistically
significant activity was the analogue VIII, having the Tle-LeuLeu sequence in the central part of the molecule compared to
the Leu-Leu-Leu of the active analogue des-Leu-PAGA (V); its
activity, however, was not higher than that of V [19]. From the
above results it can be concluded that C-truncated PAGA
analogues are not active, irrespective of the number of
leucines present, and that replacement of Leu by Tle usually
results in analogues with reduced biological activity. The
synthesis of analogues of this type was thus discontinued.
In the HN analogues subsequently studied, replacement of
cysteine by arginine and D-serine by glycine to get des-Leu-RGPAGA (IX) preserved the activity, however without enhancing
it compared to the already known activity of des-Leu-PAGA
(V). Increasing the number of leucines in the sequence
(analogues X and XI) did not improve the activity either
(Fig. 1b and c).
Finally the activity of the recently described humanin
derivative colivelin (XII) [8,9,21,10,3,31] and its des-Leu- and
plus-Leu-analogues (XIII and XIV, respectively), was tested
under our experimental conditions. Colivelin showed the
highest activity in our test, as anticipated from the literature
data on its in vivo neuroprotective activity in animal models [8].
Interestingly, the des-Leu-colivelin analogue showed similarly
high activity. As can be seen in Fig. 1c and d, both peptides
prevented deterioration of the behavior due to QNB application. Comparison of the activity of RG-PAGA (X) to that of
colivelin (XII) and the activity of des-Leu-RG-PAGA (IX) to that
of des-Leu-colivelin (XIII), underlines the positive effect of the
presence of the activity-dependent neurotrophic factor
(ADNF-9) [8] for neuroprotective action. The presence of a
fifth leucine in the plus-Leu-colivelin analogue cancelled the
positive effect of the chimeric peptide, a fact that was not seen
with the plus-Leu-RG-PAGA. Overall, our results with the
colivelin and RG-PAGA analogues can lead to the assumption
that reducing the number of successive leucines in the central
part of the molecule from four to three maintains biological
activity, while increasing this number to five does not
improve, or even, deteriorates activity. This is favorable from
the chemical synthesis point of view, as, to our experience, the
four- and, even more, the five-leucine series of humanin
analogues present difficulties in purification.
In conclusion, some of the peptides tested in this work, and
especially colivelin (XII) and des-Leu-colivelin (XIII), have a
statistically significant antiamnesic effect exhibited by reversing memory impairment induced by QNB. Considering that
drug injections were given before the test, the reported
findings are interpreted as effects on memory retrieval. These
active peptides demonstrated the potential to modulate the
cholinergic system and improve both short-term and longterm memory retrieval in rodents. Their central effect was
exerted after i.p. administration, a route that was also used in
our previous work with the active des-Leu-PAGA (V) [19]. This
is a significant result, since activity was not induced after
direct, local i.c.v. administration. Whether these active
humanin analogues act directly on the CNS, after crossing
the blood–brain barrier, or indirectly by activating peripheral
pathways, remains to be elucidated. No direct evidence
concerning the brain availability of i.c.v., i.p., or i.n.
[20,19,31] administered humanin or other peptide of the
humanin family or their ability to cross the blood brain barrier
has been published so far. Such data would contribute a lot to
the elucidation of the mode of action of this class of
neuroprotective peptides that offer hope for the treatment
of the cognitive deficits associated with AD and other
neurodegenerative diseases.
Acknowledgements
The work was supported by the Grant Agency of Czech
Republic No. 305/03/1100.
The authors wish to acknowledge financial support by the
Czech-Greek Joint Research and Technology Programme 2005–
2007, grant No. 188-g, and MSMT Czech Republic, grant No. ME
872.
The authors also thank Mrs. Eva Reslova for performing the
behavioral test.
references
[1] Afifi AA, Azen SP, editors. Statistical analysis and computer
oriented approach. 2nd ed., New York: Academic Press;
1979. p. 442–5.
[2] Albanus L. Studies on central and peripheral effects of
anticholinergic drugs. FOA Rep 1970;4:1–17.
[3] Arakawa T, Niikura T, Arisaka F, Kita Y. Activity-dependent
neurotrophic factor, ADNF, determines the structure
characteristics of colivelin, a fusion protein of ADNF9 and
humanin analog. J Pep Sci 2008;14:631–6.
[4] Baldini C, Formaggio F, Crisma M, Broxterman QB, Kaptein
B, Toniolo C. Preferred conformation of tert-leucine
Peptides. In: Fridkin M, Flegel M, Gilon C, Slaninova J,
editors, Peptides 2004. Proceedings of the third IPS and 28th
EPS, Prague 2004. Israel: KENES; 2005. p. 995–6.
peptides 29 (2008) 1982–1987
[5] Bartus AT, Dean RL, Beer B, Lippa AS. The cholinergic
hypothesis of geriatric memory dysfunction. Science
1982;217:408–17.
[6] Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease.
Lancet 2006;368:387–403.
[7] Bureš J, Burešová O, Huston J, editors. Techniques and basic
experiments for the study of brain and behavior.
Amsterdam: Elsevier; 1979. p. 147–50.
[8] Chiba T, Yamada M, Hashimoto Y, Sato M, Sasabe J, Kita Y,
et al. Development of a femtomolar-acting humanin
derivative named colivelin by attaching activity-dependent
neurotrophic factor to its N terminus: characterization of
colivelin-mediated neuroprotection against Alzheimer’s
disease-relevant insults in vitro and in vivo. J Neurosci
2005;25:10252–61.
[9] Chiba T, Yamada M, Sasabe J, Terashita K, Aiso S, Matsuoka
M, et al. Colivelin prolongs survival of an ALS model mouse.
Biochem Biophys Res Commun 2006;343:793–8.
[10] Chiba T, Nishimoto I, Aiso S, Matsuoka M. Neuroprotection
against neurodegenerative diseases: development of a
novel hybrid neuroprotective peptide colivelin. Mol
Neurobiol 2007;35:55–84.
[11] Daiello LA. Current issues in dementia pharmacology. Am J
Manage Care 2007;13:S198–202.
[12] Evangelou A, Zikos C, Livaniou E, Evangelatos GP. Highyield, solid-phase synthesis of humanin, an Alzheimer’s
disease associated, novel 24-mer peptide which contains a
difficult sequence. J Pept Sci 2004;10:631–5.
[13] Hashimoto Y, Niikura T, Tajima H, Yasukawa T, Sudo H, Ito
Y, et al. A rescue factor abolishing neuronal cell death by a
wide spectrum of familial Alzheimer’s disease genes and
abeta. Proc Natl Acad Sci USA 2001;98:6336–41.
[14] Hashimoto Y, Ito Y, Niikura T, Shao Z, Hata M, Oyama F,
et al. Mechanisms of neuroprotection by a novel rescue
factor humanin from Swedish mutant amyloid precursor
protein. Biochim Biophys Res Commun 2001;283:460–8.
[15] Kariya S, Takahashi N, Ooba N, Kawahara M, Nakayama H,
Ueno S. Humanin inhibits cell death of serum-deprived
PC12h cells. Neuroreport 2002;13:903–7.
[16] Kawasumi M, Hashimoto Y, Chiba T, Kanekura K,
Yamagishi Y, Ishizaka M, et al. Molecular mechanisms
for neuronal cell death by Alzheimer’s amyloid
precursor protein-relevant insults. Neurosignals
2002;11:236–50.
[17] Kinney HC, Panigrahy A, Rava LA, White WF. 3Dimensional distribution of [H3] quinuclidinyl benzilate
binding to muscarinic cholinergic receptors in the
developing human brain-stem. J Comp Neurol
1995;362:350–67.
1987
[18] Krejčová G, Kassa J, Vachek J. Effects of atropine and the
oxime HI-6 on low-level sarin-induced alteration of
performance of rats in a T-maze. Acta Med (Hradec
Králové) 2002;45:107–10.
[19] Krejčová G, Patočka J, Slaninová J. Effect of humanin
analogues on experimentally induced impairment of
spatial memory in rats. J Pept Sci 2004;10:636–9.
[20] Mamiya T, Ukai M. [Gly14]-Humanin improved the learning
and memory impairment induced by scopolamine in vivo.
Br J Pharmacol 2001;134:1597–9.
[21] Matsuoka M, Hashimoto Y, Aiso S, Nishimoto I. Humanin
and colivelin: neuronal-death-suppressing peptides for
Alzheimer’s disease and amyotrophic lateral sclerosis. CNS
Drug Rev 2006;12:113–22.
[22] Mezey Sz, Székely AD, Bourne RC, Kabai P, Csillag A.
Changes in binding to muscarinic and nicotinic cholinergic
receptors in the chick telencephalon, following passive
avoidance learning. Neurosci Lett 1999;270:75–8.
[23] Nieoullon A. Alzheimer’s disease: neurobiological advances
supporting proposals for new therapeutical approaches. J
Appl Biomed 2004;2:123–30.
[24] Niikura T, Hashimoto Y, Tajima H, Nishimoto I. Death and
survival of neuronal cells exposed to Alzheimer’s insults. J
Neurosci Res 2002;70:380–91.
[25] Niikura T, Tajima H, Kita Y. Neuronal cell death in
Alzheimer’s disease and neuroprotective factor humanin.
Curr Neuropharmacol 2006;4:139–47.
[26] Patočka J, Slaninova J, Kunešová J. Neuroprotective
peptides as drug candidates against Alzheimer’s disease. J
Appl Biomed 2005;3:67–73.
[27] Piccioto MR, Zoli M. Nicotinic receptors in aging and
dementia. J Neurobiol 2002;53:641–55.
[28] Schneck HJ, Rupreht J, Waldvogel HH, Hundelshausen BV.
The central anticholinergic syndrome. J Anaesth Clin
Pharm 1995;11:267–73.
[29] Tajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y, Terashita
K, et al. Evidence for in vivo production of humanin peptide,
a neuroprotective factor against Alzheimer’s diseaserelated insults. Neurosci Lett 2002;324:227–31.
[30] Terashita K, Hashimoto Y, Niikura T, Tajima H, Yamagishi
Y, Ishizaka M, et al. Two serine residues distinctly regulate
the rescue function of humanin, an inhibiting factor of
Alzheimer’s disease-related neurotoxicity: functional
potentiation by isomerization and dimerization. J
Neurochem 2003;85:1521–38.
[31] Yamada M, Chiba T, Sasabe J, Terashita K, Aiso S, Matsuoka
M. Nasal Colivelin treatment ameliorates memory
impairment related to Alzheimer’s disease.
Neuropsychopharmacology 2008;33:2020–32.