Attenuating amyloid-ß mediated neurodegenera-tion is of major therapeutic consideration in thepotential treatment of Alzheimer disease.Previously, we found that a high dietary consump-tion of retinoic acid was associated with a reducedincidence of Alzheimer disease. Therefore, in thisstudy, we investigated whether amyloid-ß mediatedcell death in primary hippocampal neurons couldbe prevented by retinoic acid isomers. Our resultssuggest that retinoic acid isomers, including all-trans retinoic acid, 9-cis retinoic acid, and 13-cisretinoic acid, may play an important role in pro-tecting neurons from amyloid-ß-induced cell death.Retinoic acid may therefore afford a novel thera-peutic mechanism for the treatment and preventionof Alzheimer disease.

Keywords: Apoptosis; all-trans Retinoic acid; 9-cis Retinoicacid; 13-cis Retinoic acid; Amyloid-ß; Hippocampal neurons

INTRODUCTION

The major pathological characteristic of Alzheimer dis-ease (AD) is the accumulation of a 40-43-amino acidpeptide, amyloid-ß (Aß), which constitutes the majorproteinaceous element of senile plaques found in ADand in Down syndrome (Glenner and Wong, 1984;Masters et al., 1985). Aß is derived from the proteolyt-ic processing of the amyloid-ß protein precursor(AßPP), a type I transmembrane glycoprotein that isexpressed in a variety of cells during normal cellularmetabolism (Haass et al., 1992). Since cell death is ahallmark feature of neurodegeneration in AD and Aß

can induce neuronal cell death in vitro and in vivo (Looet al., 1993; Paradis et al., 1996), many investigatorshave proposed Aß as a key mediator of disease patho-genesis (Smith et al., 1998; Selkoe, 2001) and consid-erable efforts are being made to develop agents toattenuate Aß toxicity.

In a previous nutritional analysis of risk factors forAD, we found that a diet high in retinoic acid (RA), anactive metabolite of vitamin A, was associated with areduced risk of disease (Smith et al., 1999). RA regu-lates a wide range of biological processes includingcell proliferation, differentiation, and morphogenesis(De Luca, 1991) and it is notable that derrangements inthese processes are all reported in AD (Raina et al.,2000; Obrenovich et al., 2004). Therefore, RA couldhave a multitude of beneficial pleotrophic effects inpatients with disease. To begin to address this in thepresent study, since Aß is considered a primary targetfor disease management, we determined whether RAisomers protect primary cultures of hippocampal neu-rons from Aß-induced neurodegeneration and therebymight be of therapeutic value.

MATERIALS AND METHODS

Minumum Essential Medium with Earle's salt (MEM)and fetal bovine serum were obtained from GibcoBRL. Bisbenzimide (Hoechst 33258), poly-L-lysine(MW 70-150 kDa), all-trans retinoic acid, 9-cis retinoicacid, 13-cis retinoic acid were purchased from Sigma.Poly-L-lysine was dissolved in borate buffer (pH 8.4).The effects of Aß are mediated by amino acid residues25-35 of the full-length peptide (Mattson et al., 1992),which is directly toxic to neurons (Pike et al., 1993)

F.P. Graham Publishing Co.

Neurotoxicity Research, 2005, VOL. 7(3). pp. 243-250

Retinoic Acid Isomers Protect Hippocampal Neurons From Amyloid-β Induced NeurodegenerationMEHMET SAHINa, SIBEL BERKER KARAÜZÜMb, GEORGE PERRYc, MARK A. SMITHc,* and YAKUPALICIGÜZELa

Akdeniz University, Faculty of Medicine, aDepartment of Biochemistry and bDepartment of Medical Biology and Genetics,Dumlupinar Boulevard, 07070, Arapsuyu-Antalya, Turkey; and cInstitute of Pathology, Case Western Reserve University,Cleveland, OH, USA. mark.smith@case.edu

(Received 09 July 2004; Revised 16 December 2004; In final form 16 December 2004)

*Corresponding authors. E-mail: mark.smith@case.edu. or yakup@akdeniz.edu.trISSN 1029 8428 print/ ISSN 1476-3524 online. © 2005 FP Graham Publishing Co., www.NeurotoxicityResearch.com

and also increase the vulnerability of neurons to otherinsults (Paradis et al., 1996). Aß25-35 (Sigma) was dis-solved in sterile deionized water and stored in at 20°C.To obtain the neurotoxic form of Aß25-35, the peptidesolution was placed in an incubator at 37°C for 7 days(Pike et al., 1993).

Hippocampal cell cultures were prepared as previ-ously described (Mattson et al., 1995) with minor mod-ifications. All procedures were performed under sterileconditions. Briefly, cultures were prepared from 1-2-day-old Wistar rats. After the brains were removed,hippocampi were rapidly removed by stereomicroscop-ic dissection and incubated in 0.25% trypsin solution inHanks' Balanced Salt Solution without calcium andmagnesium (Gibco) at 37°C for 20 min. After enzy-matic dissociation, cells were triturated with a Pasteurpipette flamed to half its original opening size. Theresulting cell suspension was seeded onto a poly-L-lysine (10 µg/ml) coated petri dish to a density of 2 x106 cells/35 mm diameter petri dish. The cell cultureswere maintained in MEM supplemented with 10% fetalbovine serum and penicillin-streptomycin at 37°C and5% CO2 in a humidified atmosphere. The medium waschanged every two days and the cells were used forexperiment on day 8 after seeding. Cultured cells weretreated with cytosine arabinoside (5 mg/l) for 24 h(between 2 and 3 days after plating) to inhibit the pro-liferation of non-neuronal cells. Thus, approximately90% of total cells were neurons (FIG. 1). At the time ofthe experiment, cells were first changed into low serum(5%) medium, then 30 µM Aß25-35 (Xia et al., 2003),and different concentrations of RA isomers were addedsimultaneously for 24 h.

RA isomers (all-trans RA:t-RA; 9-cis-RA:9-cRA;and 13-cisRA:12-cRA) were prepared as stock solu-tions in ethanol and appropriate dilutions were thenadded to the culture medium. The final concentrationsof ethanol in all experiments were < 0.01%. The medi-um supplied with low serum (5%) and ethanol (<0.01%) were also applied to control groups.

To determine the percentage of apoptotic neurons, cellswere stained with the DNA fluorochrome Hoechst 33258(1 µg/ml) for 20 min at RT. Thereafter, nuclear morphol-ogy was observed under a fluorescence microscope.Viable cells showed a normal sized nucleus with normalchromatin. In contrast, neurons with condensed chro-matin (visible as an intense fluorescence) at the nuclearperiphery or nuclear fragmentation with reduction ofnuclear size were considered as typical features of apop-tosis. Data were expressed as the percentage of apoptot-ic cells/total cell number of 10 randomized subfields ofeach separate culture petri dish (Krohn et al., 1999).

RESULTS

Rat hippocampal neurons grown for 8 days wereexposed to Aß25-35 for 24 h, and apoptotic cells weredetermined by Hoechst 33258 labelling and morpho-logical criteria (FIG. 2). While neurons in control medium for 24 h had 16%apoptotic nuclei, Aß increased the percentage of apop-totic neurons from 16% in controls to 81% (p <0.01)(Table I and FIG. 3). Figure 1 shows representativephotomicrographs of these groups.

t-RA, 9-c-RA and 13-c-RA at concentrations of 0.01,0.1 and 1 µM, were all found to dose-dependentlydecrease the percentage of apoptotic neurons (*p <0.01 for all concentrations). In t-RA, 9-c-RA and 13-c-RA groups, isomers at 1 µM were more effective thanthat at 0.1 µM; similarly, isomers at 0.1 µM were moreeffective than at 0.01 µM (Table I and FIG. 3). Theresults of the present study show that the most effectivedose was 1 µM for all isomers. t-RA, 9-c-RA and 13-c-RA in this concentration reduced the number of apop-totic nuclei from 81% in Aß-treated group to 20%, 23%and 24%, respectively (p <0.01 for all groups) (Table Iand FIG. 3).

t-RA, 9-c-RA and 13-c-RA in concentration of 0.1µM reduced the number of apoptotic nuclei from 81%in Aß-treated group to 25%, 26% and 28%, respective-ly (p <0.01 for all groups) (Table I and FIG. 3).

t-RA, 9-c-RA and 13-c-RA in a concentration of 0.01µM reduced the number of apoptotic nuclei from 81%

M. SAHIN et al.244

in Aß-treated group to 32%, 35% and 38%, respective-ly (p <0.01 for all groups) (Table I and FIG. 3).

DISCUSSION

While the role of Aß in the pathogenesis and progres-sion of AD has not yet been fully determined, it is clearthat deposits of insoluble Aß are found in plaques in the

brains, particularly the hippocampus, of patients withAD; and that insoluble Aß can induce apoptotic neu-ronal cell death in vitro (Selkoe, 1990; Pike et al.,1991). In this report, we described the anti-apoptoticproperties of t-RA, 9-c-RA and 13-c-RA in hippocam-pal cells exposed to Aß. Specifically, Aß-mediated celldeath of hippocampal neurons exposed to Aß25-35 wassignificantly attenuated, in a dose-dependent manner,

RETINOIC ACID PROTECTION AGAINST Aβ NEUROTOXICITY 245

A B

C D

EF

G

FIGURE 1 Photomicrographs of hippocampal neurons. Cultured cells were treated with cytosine arabinoside (5 mg/l) for 24 h to reduceastrocytes and glial proliferation. Neurons at this stage (A), treated with Aß25-35 plus 9-c-RA (B), x40 magnification. Neurons exposed for24 h to control medium containing low serum plus ethanol (C), medium containing Aß25-35 30 µmol/l (D), medium containing Aß25-35 30µmol/l and t-RA (E), 9-c-RA (F), and 13-c-RA (G), x10 magnification.

by RA and RA isomers. Agents that attenuate Aß-medi-ated cell death are potentially of therapeutic impor-tance since Aß toxicity has been suggested to beresponsible for at least some of the neurodegenerationobserved in the vulnerable regions of the brain in AD(Selkoe, 2001) possibly via apoptotic mechanisms(Loo et al., 1993; Paradis et al., 1996) although thisremains controversial in vivo (Raina et al., 2001).

Aß is thought to exert its neurotoxicity via severalsignaling mechanisms. Aß can influence neuronsthrough activation of signaling pathways, and free-rad-ical accumulation. For example, it was reported that inneurons exposed to Aß, activated JNK (c-Jun N-termi-nal kinase) is required for the phosphorylation and acti-vation of the c-Jun transcription factor, which in turnstimulates the transcription of several key target genes,including the death-inducer Fas ligand (Morishima etal., 2001). Importantly, such signal transduction cas-cades are also activated in vivo in neurons in the ADbrain (Zhu et al., 2001a,b; 2003; 2004). Also, it hasbeen reported that Aß25-35 and Aß1-42 induce the tyrosinephosphorylation of numerous neuronal proteins,

including tau and microtubule-associated protein 2c,activating the Src family tyrosine kinase (Williamsonet al., 2002). In addition, it was shown that Aß inducestau phosphorylation by the progressive and sustainedactivation of the MAPK in mature hippocampal neu-rons (Rapoport and Ferreira, 2000) and again thesekinases are also found in neurons in AD cases but notage-matched controls (Zhu et al., 2001b).

Cyclin-dependent kinase-5 and its neuron-specificactivator p35 are required for neurite outgrowth andcortical lamination. Proteolytic cleavage of p35 pro-duces p25, which accumulates in the brain of patientswith AD. Application of Aß induces the conversion ofp35 to p25 in cortical neurons, leading to the activationof cdk-5 and tau phosphorylation (Patrick et al., 1999;Lee et al., 2000).

t-RA and its stereoisomer 9-c-RA, two active meta-bolites of vitamin A, are known to regulate a broadrange of biological processes, including vertebratedevelopment, growth, and differentiation (Chambon,1995; Napoli, 1996). Recent studies suggest thatretinoids may play important roles in the physiological

M. SAHIN et al.246

FIGURE 2 Flourescent micrographs of cultured rat hippocampal neurons exposed for 24 h to control medium; mitotic division is seen atthe center of figure (indicated with arrow) (A), medium containing Aß25-35 30 µmol/l (B), medium containing Aß25-35 30 µmol/l and t-RA(C), 9-c-RA (D), and 13-c-RA (E). Hoechst 33258 fluorescence staining, x40. Condensed or fragmented apoptotic nuclei are indicated witharrows in the some area of the figures.

E

D

C

A

B

and pathophysiological functions of the hippocampus.Indeed, various forms of retinoid receptors are found inthe central nervous system including the hippocampus(Zetterstrom et al., 1999). Moreover, as documented bya number of studies in various cell systems (Nabeyratet al., 1998; Chung et al., 2000), RA appears to affectcell-cycle progression by acting mainly on regulatoryproteins involved in the G1 phase and in the G1/S-phase transition (Langenfeld et al., 1996; Seewaldt etal., 1997). It was previously demonstrated that RAmediated its effect on the proliferative response of oxi-dant-exposed cells through an inhibition of p21CIP1

expression, which resulted in a reduced interaction ofp21CIP1 with cyclin E-CDK2 complexes (Nabeyrat etal., 2001). Notably, we and others find significant andprofound alterations in numerous compounds of thecell cycle machinery in AD (reviewed in Raina et al.,2000; Ogawa et al., 2003) raising the possibility thatRA exerts a protective effect in AD, at least partiallythrough attenuation of cell cycle alterations.

It is well established that RA effects are mediatedthrough nuclear RA receptors (RARs) and retinoid Xreceptors (RXRs). These receptors act as DNA-bindingproteins, which can activate or repress transcription ofa multitude of target genes. It has been shown that thegene coding for p21CIP1 possesses an RAR/RXR-responsive element in its promoter (Liu et al., 1996). Inour study, RA isomers may also use such a responsepathway. So the cells treated with RA plus Aß may pro-liferate and there may be a decrease in the proportionof apoptotic cells.

In addition to the role of Aß on signaling patways,oxidative stress has been implicated in many aspects ofaging and neurodegenerative diseases, including AD(Smith et al., 1992; Coyle and Puttfarcken, 1993), andit is notable that the neurotoxicity of Aß also appears toinvolve oxidative stress (Rottkamp et al., 2001; Smithet al., 2002). That Aß may serve a role in modulatingredox homeostasis (Nunomura et al., 2001) and couldeven be an antioxidant (Smith et al., 2002) is consistentwith both the in vitro and in vivo properties of Aß.Indeed, most antioxidants are also prooxidants depend-ing on the context. As such, it is not suprising that Aßgenerates free radical in solution (Hensley et al., 1994)and induces peroxide accumulation in cultured neurob-lastoma cells (Behl et al., 1994) and primary hip-pocampal neurons (Goodman and Mattson, 1994).Moreover, Aß toxicity can be blocked by antioxidants(Behl et al., 1994; Goodman and Mattson, 1994;Hensley et al., 1994). Importantly, retinoids possessantioxidant activity (Packer, 1990). For example, RAinhibits lipid peroxidation by scavenging lipid peroxylradicals (Das, 1989; Samokyszyn and Marnett, 1990).The cytoprotective action of RA against H2O2 might besimply via scavenging cytotoxic reactive oxygenspecies (ROS). Alternatively, RA may induce endoge-nous antioxidant enzymes since t-RA up-regulatescatalase activity and the reduced form of glutathione(GSH) content via transcriptional upregulation of cata-lase and γ-glutamyl-cysteine synthetase, the limitingenzyme of GSH synthesis (Moreno-Manzano et al.,1999). Our results are in accordance with the findings

RETINOIC ACID PROTECTION AGAINST Aβ NEUROTOXICITY 247

FIGURE 3 t-RA protected the cultured neurons from apoptosis induced by Aß25-35 (30 µmol/l). Results are the mean ± SD of four indi-viduals. **P <0.01 vs Aß25-35 (student's t-test). C: control; A: Aß25-35; t-RA: all-trans retinoic acid. 9-c-RA protected the cultured neuronsfrom apoptosis induced by Aß25-35 (30 µmol/l). Results are the mean ± SD of four individuals. **P <0.01 vs Aß25-35 (student's t-test). C:control; A: Aß25-35; 9-c-RA: 9-cis retinoic acid. 13-c-RA protected the cultured neurons from apoptosis induced by Aß25-35 (30 µmol/l).Results are the mean ± SD of four individuals. **P <0.01 vs Aß25-35 (student's t-test). C: control; A: Aß25-35; 13-c-RA: 13-cis retinoic acid.

of Ahlemeyer and Krieglstein (1998; 2000), whodemonstrated that tocopherol and RA, which have beenpreviously described to have anti-apoptotic and antiox-idant properties were able to inhibit staurosporine-induced apoptosis and to reduce mitochondrial ROSproduction in hippocampal neurons.

In addition to antioxidant properties, RA has beenconsidered as a potential therapeutic agent for malig-nant diseases, especially for the treatment of leukemia(Schule et al., 1991), due to its pharmacological poten-tial to induce growth arrest and cellular differentiation(Packer, 1990). Notably, we and others find strikingsimilarities between processes involved in AD andthose involved in oncogenic events (Zhu et al., 2000;2001b). RA may inhibit apoptosis of T cells, leukemiccells, and hematopoietic cells (Iwata et al., 1992; Yanget al., 1993; Zauli et al., 1995; Ketley et al., 1997).Using Aß as a trigger, the present report provides novelevidence for the anti-apoptotic potential of RA isomers.

Finally, it is of note that it was shown that there isdefective retinoid transport and function in late onsetAD (Goodman and Pardee., 2003). This study supportsour idea that RA supplementation may play a protec-tive role in AD. RA would be a potential target fordrugs aimed at stopping or at least slowing AD devel-opment. The drugs could induce enzymes that increaseRA synthesis, in particular, in the hippocampus andother areas involved in AD pathology. However, RA istoxic at high concentrations. Thousands of RA analogshave therefore been synthesized in efforts to diminishtoxicity relative to efficacy and ligand specificity.Some of these might be effective against AD. A lesstoxic alternative might be with retinyl esters, bound tochylomicrons (Goodman and Pardee., 2003).

In conclusion, our study further suggests that the pro-gression of Aß toxicity in AD may be reduced by RAsupplementation and that RA may therefore be of ther-apeutic potential in aging and age-related illness.Certainly, such a notion is consistent with a studyshowing a decreased incidence of AD in individualswith high RA dietary intake (Smith et al., 1999).

Acknowledgements

This work was supported by Akdeniz UniversityResearch Fund (Project Number: 21.01.0122.10).

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