Anti-apoptotic action of anti-Alzheimer drug, TV3326 [(N-propargyl)-(3R)-aminoindan-5-yl]-ethyl methyl carbamate, a novel choliAcknowledgementsReferencesAnti-apoptotic action of anti-Alzheimer drug, TV3326 [(N-propargyl)-(3R)-aminoindan-5-yl]-ethyl methyl carbamate, a novel cholinesterase-monoamine oxidase inhibitorWakako Maruyamaa, Marta Weinstockb, Moussa B.H. Youdimc, Masayo Nagaia, Makoto Naoid,*aLaboratory of Biochemistry and Metabolism, Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Aichi, JapanbDepartment of Pharmacology, Hebrew University Hadassah School of Medicine, Jerusalem, IsraelcEve Topf and NPF Centers, Technion-Faculty of Medicine, Haifa, IsraeldDepartment of Brain Sciences, Institute of Applied Biochemistry, Yagi Memorial Park, Mitake, Gifu 505-0116, JapanReceived 19 August 2002; received in revised form 5 February 2003; accepted 7 February 2003AbstractThe anti Parkinson drug, rasagiline [R-(þ )-N-propargyl-1-aminoindan], an inhibitor of type B monoamine oxidase, has been shown tosuppress apoptosis induced by neurotoxins and oxidative stress. A series of novel propargylaminoindans with a carbamate moiety to inhibitcholinesterase were developed from phamacophore of rasagiline to protect or rescue deteriorated neurons in Alzheimer’s and Lewy Bodydisease and provide a beneficial effect on the cognitive deficits. Rasagiline analogues were found to protect dopaminergic SH-SY5Y cellsagainst apoptosis induced by peroxynitrite donor. SIN-1. TV3326, [(N-propargyl)-(3R)-aminoindan-5-yl]-ethyl methyl carbamate, was aseffective as rasagiline in preventing apoptosis, followed by its S-enantiomer, TV3279. The anti-apoptotic-neuroprotective activity was shownto reside in the propargylamine and not the carbamate moiety. This resulted in stabilization of the mitochondrial membrane potential, thecollapse of which initiates the apoptotic cascade.q 2003 Elsevier Science Ireland Ltd. All rights reserved.Keywords: Propargylamines; Anti-apoptotic activity; Mitochondrial membrane potential; Monoamine oxidase inhibitor; Cholinesterase inhibitor; Parkinson’sdisease; Alzheimer’s disease; Lewy Body disease; Peroxynitrite; Rasagiline; TV3326Neurodegenerative disorders, such as Parkinson’s (PD) andAlzheimer‘s disease (AD), are characterized by progressivecell death of selective neurons in the brain. Apoptosis isconsidered to be a common type of neuronal cell death inneurodegenerative diseases that may be induced by variousenvironmental and genetic factors. The apoptotic cascade isactivated by tightly controlled step-wise processes and hasbeen proposed to be a target of neurorescue or neuropro-tective strategies [15].The anti-Parkinson drug, rasagiline[N-propargyl-(1R)-aminoindan] [5], a selective irreversiblemonoamine oxidase (MAO)-B inhibitor [19] has neuropro-tective activity against death in cultured hippocampal, PC-12 and SH-SY5Ycells [4,7,11,20] and in animal models ofhead trauma [6], and MPTP induced neurotoxicity [14].Thesuppression by rasagiline of the apoptotic process inducedby an endogenous neurotoxin, N-methyl(R)salsolinol and adonor, SIN-1 was confirmed in human dopaminergicneuroblastoma SH-SY5Y cells [8,9]. Structure-activitystudies have indicated that the propargyl moiety is essentialfor the anti-apoptotic function of cyclic benzyl-(rasagiline)[8] and aliphatic-[N-(2-heptyl)-N-propargylamine] propar-gylamines [9]. In addition, the anti-apoptotic activity ofrasagiline is independent of inhibition of MAO-B, since itsS-isomer, TVP1022, lacking in MAO inhibitory activity stillprotects SH-SY5Y and PC12 cells which do not expressMAO-B [7,11,18].A series of analogues were synthesized with a carbamatecholinesterase inhibitory moiety in the aminoindan structureof rasagiline with the purpose of preserving its neuropro-tective activity [6,15,17,18] and also to inhibit acetylchol-inesterase [acetylcholine acetylhydrolase, EC 3.1.1.7, ChE]to increase cholinergic transmission. The deficits incholinergic activities are closely related to clinical symp-toms in AD and Lewy Body disease and cholinesteraseinhibitors, such as rivastigmine, have been shown to have0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.doi:10.1016/S0304-3940(03)00211-8Neuroscience Letters 341 (2003) 233–236www.elsevier.com/locate/neulet* Corresponding author. Tel.: þ 81-574-67-5500; fax: þ 81-574-67-5310.E-mail address: [email protected] (M. Naoi).beneficial effects in such subjects [2]. TV3326, [(N-propagyl-(3R)aminoindan-5-yl)-ethyl methyl carbamate](Fig. 1), inhibits acetylcholinesterase (ChE) and is brainselective type A and B MAO inhibitor and improvesmemory impairments in scopolamine treated rats. Its S-isomer, TV3279, which is also ChE inhibitor (Fig. 1) has noMAO inhibitory activity [16,17] but also has similar actionin scopolamine impairment test. These compounds retainedthe neuroprotective activities of rasagiline in partiallydifferentiated PC-12 cells deprived of serum and NGF andin vivo [16,18,20]. Furthermore, recently they have beenshown to process amyloid precursor protein in vitro and invivo by a PKC and MAP kinase dependent reaction andrelease the neuroprotective anti-apoptotic soluble amyloidprecursor protein alpha [18].In this paper, neuroprotective activities of TV3326, its S-isomer, TV3279, and related compounds were examined fortheir potential protection against apoptosis and the fall inmitochondrial membrane potential (DCm) associated withapoptosis induced by the peroxynitrite-generating agent,SIN-1 [N-morpholino sydnonimine] in dopaminergic neu-roblastoma SH-SY5Y cells.Rasagiline and derivatives (Fig. 1) were kindly donatedby Teva Pharmaceutical (Netanya, Israel). Hoechst 33342and Rhodamin 123 were purchased from Molecular Probes(Eugene, OR, USA); SIN-1 from Dojindo (Kumamoto,Japan); propidium iodide (PI) from Sigma (St. Louis, MO,USA); other reagents were from Nacalai Tesque (Kyoto,Japan).SH-SY5Y cells were incubated with 0.01–10 mMofpropargylamine derivatives for 20 min, then cultured for 18h in the presence of 250 mM SIN-1, and the morphologicalchanges in the cells were observed by phase-contrast andfluorescence microscopy after staining with PI and Hoechst33342 [8]. PI (at the final concentration of 10 mM) andHoechst 33342 (10 mM) was added to the medium and thecells were incubated for 30 min at 37 8C. Cells stained withPI were observed fluorometrically (emission, above 580 nm;excitation, 520–550 nm)