Neurobiology of Aging xxx (2014) 1e14

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Neuroprotective and neurorestorative activities of a novel iron chelator-brain selective monoamine oxidase-A/monoamine oxidase-B inhibitor in animal models of Parkinson’s disease and aging Orit Bar-Am a, Tamar Amit a, Lana Kupershmidt a, Yuval Aluf a, Danit Mechlovich a, Hoda Kabha a, Lena Danovitch a, Vincent R. Zurawski a, Moussa B.H. Youdim a, b, *, Orly Weinreb a, b a b

Varinel Pharmaceuticals Ltd, New Southern Industrial Park, Yokneam, Israel Eve Topf Center, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 July 2014 Received in revised form 19 October 2014 Accepted 19 October 2014

Recently, we have designed and synthesized a novel multipotent, brain-permeable iron-chelating drug, VAR10303 (VAR), possessing both propargyl and monoamine oxidase (MAO) inhibitory moieties. The present study was undertaken to determine the multiple pharmacological activities of VAR in neurodegenerative preclinical models. We demonstrate that VAR affords iron chelating/iron-induced lipidperoxidation inhibitory potency and brain selective MAO-A and MAO-B inhibitory effects, with only limited tyramine-cardiovascular potentiation of blood pressure. The results show that in 6hydroxydopamine rat (neuroprotection) and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse (neurorescue) Parkinson’s disease models, VAR significantly attenuated the loss of striatal dopamine levels, markedly reduced dopamine turnover, and increased tyrosine-hydroxylase levels. Furthermore, chronic systemic treatment of aged rats with VAR improved cognitive behavior deficits and enhanced the expression levels of neurotrophic factors (e.g., brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and nerve growth factor), Bcl-2 family members and synaptic plasticity in the hippocampus. Our study indicates that the multitarget compound VAR exerted neuroprotective and neurorestorative effects in animal models of Parkinson’s disease and aging, further suggesting that a drug that can regulate multiple brain targets could be an ideal treatment-strategy for age-associated neurodegenerative disorders. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease Aging Iron chelation Monoamine oxidase inhibition Neurotrophic factors Neurorestoration

1. Introduction Accumulating evidence have demonstrated that increased monoamine oxidase (MAO)-B activity, excessive iron accumulation, and reduced antioxidant activities in the brain are essential pathogenic factors in neurodegenerative diseases (such as, Parkinson’s disease [PD], Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington disease, and Friedreich ataxia) (Youdim and Buccafusco, 2005; Zecca et al., 2004). In these neurodegenerative

* Corresponding author at: Eve Topf Center, Faculty of Medicine, Technion-Israel Institute of Technology, P.O.Box. 9697, 31096 Haifa, Israel. Tel.: þ972 4 8295290; fax: þ972 4 8513145. E-mail address: [email protected] (M.B.H. Youdim). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.10.026

disorders, as well as in the regular aging, iron accumulation has been observed in specific brain regions (Jellinger, 1999; Jellinger et al., 1993; Zecca et al., 2004). For example, studies have shown that iron concentrations are significantly elevated in parkinsonian substantia nigra pars compacta and within the melanized dopamine (DA) neurons (Gerlach et al., 2006; Gotz, 2004; Zecca et al., 2004). Similar results have also been reported in the 6hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) animal PD models (Gerlach et al., 2000; He et al., 1996; Youdim et al., 2004; Zecca et al., 2004). Evidence has shown that significant accumulation of iron in white matter tracts and nuclei throughout the brain precedes the onset of neurodegeneration and movement disorder symptoms (LaVaute et al., 2001). Indeed, it is well established that iron induces oxidative stress, because it initiates the Fenton reaction, resulting

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in increased formation of hydroxyl radicals that cause damage to DNA, proteins, lipids, and ultimately cell death associated with neurodegeneration (Jellinger, 1999; Zecca et al., 2004). Currently, simultaneous modulation of cascades of neurotoxic events, involved in the neurodegenerative process, by using a single multitarget drug is the most promising therapeutic approach for treatment of neurodegenerative disorders. The challenge of designing pluripotential and/or multitarget drugs is to combine multiple pharmacological moieties to a basic active molecule (Hopkins et al., 2006; Keith et al., 2005). We have therefore designed and synthesized a series of multitarget, nontoxic, brain permeable iron chelators with potent MAO inhibitory activity, based on chemically hybridizing the neuroprotective and MAO inhibitory moiety, N-propargyl, associated with the antiparkinsonian/MAO-B inhibitor, rasagiline (Azilect), into the 8hydroxyquinoline-containing pharmacophore of the prototype brain permeable iron chelator VK-28 (Zheng et al., 2005a, 2005b). Among these compounds, M30 and HLA20 were previously shown to exert iron-chelating potency, cytoprotective anti-apoptotic properties, and inhibition of iron-induced membrane lipidperoxidation features (Zheng et al., 2005a, 2005b). In vivo studies reported that M30 showed neuroprotective effects in animal models of PD, induced either by MPTP (Gal et al., 2005, 2010a, 2010b) or the proteasome inhibitor lactacystin (Zhu et al., 2007), Alzheimer’s disease (Kupershmidt et al., 2012b), amyotrophic lateral sclerosis (Kupershmidt et al., 2010), and aging (Kupershmidt et al., 2012a). Another multifunctional member of this series is the iron chelating/MAO inhibitory compound VAR10303 (VAR). Considering possible pharmacological differences between M30 and VAR, regarding metabolic activity, pharmacodynamics and pharmacokinetics, stability and resistance mechanisms, as well as safety and tolerability, we have characterized in the present study the iron chelating and anti-oxidative properties of VAR and analyzed its in vivo neuropharmacologic actions, in terms of MAO inhibition in the brain and periphery, levels and/or turnover of striatal monoamines and pressor response to tyramine. In addition, we sought to determine the respective neuroprotective and neurorestorative properties of VAR in the unilateral 6-OHDA-lesioned rat and MPTPmouse PD models, respectively. The potential beneficial effects of VAR were further examined on age-related alterations in rats, regarding antidepressant like-behavior and cognitive behavior, and hippocampal molecular signaling pathways. 2. Methods 2.1. Materials Monoclonal antibodies against phospho-cAMP response element-binding protein (CREB) (Ser133) CREB and B cell lymphoma-2 (Bcl-2), rabbit polyclonal antibodies against phospho(Thr202/Tyr204) and non-phospho-p44/42 extracellular signalregulated kinases (ERK1/2), Bax, and secondary antibodies were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). Monoclonal rabbit antibody against brain-derived neurotrophic factor (BDNF) was from Epitomics Inc (Burlingame, CA, USA). Polyclonal rabbit antibody against growth-associated protein (GAP)-43 was purchased from Chemicon (Tamecula, CA, USA). Mouse antisynaptophysin monoclonal antibody, rabbit anti-tyrosine hydroxylase (TH) polyclonal antibody and goat anti-mouse IgG fluorescein isothiocyanate conjugate AP132F were purchased from Millipore (Billerica, MA, USA). Donkey anti-rabbit IgG fluorescein-conjugated antibody was from Jackson ImmunoResearch Laboratories Inc (Baltimore, MD, USA). b-actin antibody, 6-OHDA, and MPTP were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Chemical synthesis of VAR The preparation of VAR (5-[2-(methyl-prop-2-ynyl-amino)ethyl]-quinolin-8-ol dihydrochloride) (Ricerca Biosciences, OH, USA) is outlined in Fig. 1. The Friedel-Crafts Acylation reaction of 8hydroxyquinoline (compound 1.1) with a-chloroacetyl chloride (compound 1.2) was carried out at room temperature (RT). Aluminum chloride (AlCl3) was added to the resulting yellow suspension in small portions over 1 hour, and next heated to 100  C for 36 hours, resulting in compound 1.3. Trifluoroacetic acid (TFA) was added to 1.3, and the reaction vessel was cooled to 0  C. Triethylsilane (Et3SiH) was added dropwise over 15 minutes. The reaction was gradually heated to 60  C, resulting in compound 1.4 after 16 hours. The product was placed in a tube and suspended in acetonitrile (MeCN), followed by addition of sodium iodide (NaI) and N-propargylamine (compound 1.5). The tube was sealed under a blanket of nitrogen and heated to 100  C for 48 hours. Hydrochloric acid (3 M in methanol) was added to the solution of VAR (compound 1.6) in dichloromethane (200 mL) and stirred at 25  C for 1 hour. High performance liquid chromatography system and liquid chromatography-mass spectrometry analyses showed >97% purity of VAR. 2.3. Animal and treatment procedures 2.3.1. Animals Male young C57/Bl/6J mice (2 months old) and male young (2 months old) and aged (18 months old) Sprague-Dawley rats were obtained from Harlan Laboratories, Inc, Israel. All procedures were carried out in accordance with the National Institutes of Health Guide for care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Technion, Haifa, Israel. For all experimental protocols, animals were weighted once a week. The average body weight was not significantly different between VARand vehicle-treated animals, in different experimental groups. 2.3.2. Experimental protocols and behavioral tests For acute drug administration studies, rats were treated with VAR (7.5, 10, and 12.5 mg/kg, subcutaneous [s.c.]). Control rats were given 0.9% saline s.c. For chronic drug administration studies, mice were treated with VAR (1.1, 3.3, and 10 mg/kg, per os [p.o.], daily) for 14 days. Control mice were given deionized water p.o. The effective doses of VAR used in all experiments were chosen, based on our previous studies with the multifunctional iron-chelating compound, M30 in animal models of neurodegeneration (Gal et al., 2005; Kupershmidt et al., 2012a). One day following the last administration of the drug, the animals were sacrificed by decapitation, and striatum, hippocampus, cerebellum, liver, and small intestine have been removed rapidly and frozen in liquid nitrogen for further analyses. 6-OHDA lesion: rats were anesthetized with ketamine and/or xylazine (70/35 mg/kg, intraperitoneal injection [i.p.]) and placed in a stereotaxic frame (Kopf, CA, USA). A heating pad maintained a constant body temperature of 37  C. The toxin 6-OHDA hydrochloride was injected into 2 locations (2  15 mg in 4 mL saline containing 0.1% ascorbic acid, at 1 mL/min infusion rate for 4 minutes) in the left striatum, using the following coordinates (in mm): AP, þ1.2; L, 2.5; DV,5; and AP, þ0.2, L, 3.8, and DV, 5 with respect to the bregma. The noradrenergic neurons were protected by injecting desipramine (10 mg/kg, s.c.), 30 minutes before the 6-OHDA injection. The rats were divided into the following groups: 6-OHDA-lesioned and treated with vehicle (0.9% saline) and 6-OHDA-lesioned and treated with VAR (10 mg/kg, s.c., 3 times weekly for 3 weeks) given 1 hour before the 6-OHDA injection. The sham-operated group was subjected to all procedures,

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Fig. 1. Chemical synthesis, iron-chelating, and lipid-peroxidation properties of VAR. (A) The synthesis procedure of VAR is detailed in Section 2.2. (B) Binding affinity of iron to VAR (Feþ3/VAR; molar ratio). (C) Iron-chelating potency of VAR. (D) Inhibition of iron-induced lipid-peroxidation of VAR in rat brain mitochondrial homogenates. Results represent the mean  SEM (n ¼ 3); *p < 0.05 versus control. Abbreviations: DFO, deferoxamine; MDA, malondialdehyde; SEM, standard error of the mean; VAR, VAR10303. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

except that saline was injected instead of 6-OHDA. Two weeks after the stereotaxic surgery, the rats were submitted to apomorphineinduced rotational test, used as a behavioral index to determine the potential neuroprotective effects of novel compounds for PD (Wree et al., 2011). The contralateral rotation induced by apomorphine (0.5 mg/kg, s.c.) was monitored a for 60 minutes, using a cylinder rotation apparatus. MPTP model: for the studies with MPTP, mice were administered with MPTP (20 mg/kg, i.p., once daily) for 4 consecutive days, followed by a further 4 days resting period (day 8) to allow for the full conversion of MPTP to its active metabolite, (1-methyl-4phenylpyridinium) (MPPþ) (Gal et al., 2010b). Control mice received 0.9% saline. At day 8, either VAR (20 mg/kg, p.o. daily) or deionized water were administered for additional 15 days. Behavioral analyses: with the aim of assessing the possible beneficial effects of VAR on cognition and depression in aging rats, behavioral studies (open-field performance [Barber et al., 1993]; object recognition [de Lima et al., 2005; Simola et al., 2008], and forced swim tests [Pirondi et al., 2005]) were performed in vehicle (0.9% saline)-treated young and aged rats and compared with VAR-treated (1 mg/kg, s.c., 4 times a week for 6 months) aged rats, as described recently (Kupershmidt et al., 2012a, 2012b).

Fe(II)-chelating potency (%) was calculated as follows: (1  [absorbance of sample at 562 nm]/[absorbance of control, at 562 nm]  100).

2.4. Iron-chelating properties

Irreversible, selective inhibition of peripheral MAO-A prevents the metabolism of tyramine (presents in food and beverage), thus resulting in increased levels of tyramine in systemic circulation, followed by the release of stored norepinephrine into peripheral adrenergic synapse, which leads to increases in blood pressure (BP) (Meck et al., 2003). For BP potentiation in response to tyramine, rats were treated with vehicle (0.9% saline) or VAR (7.5, 10, and 12.5 mg/kg, s.c.), 16 hours preceding tyramine potentiation studies and compared with the non-selective MAO-A and MAO-B inhibitor,

The binding of ferrous ions by the drug was estimated by the decreases in the maximal absorbance of the Fe(II)-ferrozine complex, as previously described (Zheng et al., 2005b). Briefly, iron precomplexed calcein (Fe-CAL) solutions (1 mM) in Hepes buffered saline (20 mM, pH 7.4) were incubated with various concentrations of VAR at RT. The fluorescence intensity (460 nm) was followed with time in Tecan Sunrise Elisa Reader (Männedorf, Switzerland).

2.5. Lipid-peroxidation assay Lipid peroxidation was assessed as previously described (Gassen et al., 1996). Briefly, homogenates of mitochondria isolated from rat brain (10 mL) were suspended in 150 mL of 25 mM Tris-HC1 (pH 7.5), containing 50 mM ascorbic acid. VAR was dissolved in water and added to the suspension. The reaction was started by the addition of 10 mL FeSO4 (from a 1.0 mM stock solution), incubated at RT for 2 hours. The incubation was stopped by the addition of 150 mL of 20% (wt/vol) trichloroacetic acid. The samples were centrifuged (12,000g, 10 minutes), and 200 mL supernatant were mixed with 500 mL of 0.5% (wt/vol) thiobarbituric acid and heated to 95  C for 30 minutes. The absorption of thiobarbituric acid derivatives was measured at 532 nm. The IC50 value was determined at the concentration of the drug required to reduce 50% of the absorbance with respect to the control. 2.6. Measurement of the blood pressure potentiation in response to tyramine

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Fig. 2. Effect of VAR treatment on MAO activity in brain and peripheral tissues. Mice received either vehicle (control) or increasing doses of VAR for 14 days. MAO-A and MAO-B activities were determined in brain, liver, and small intestine. Results are expressed as percentage inhibition of control and represent the mean  SEM (n ¼ 6e7); *p < 0.05 versus respective controls. Abbreviations: MAO, monoamine oxidase; SEM, standard error of the mean; VAR, VAR10303.

tranylcypromine, (TCP) (20 mg/kg, i.p.), administrated 1 hour before the experiment. Tyramine was administered orally at increasing concentrations (5e50 mg/kg) to provide a maximal increase in BP of more than 30 mmHg. The increase in BP potentiation in response to increasing doses of tyramine following various concentrations of VAR, compared with TCP was continuously monitored with a pressure transducer (model 50,110, Stoelting, Wood Dale, IL) connected to the carotid arterial line. 2.7. MAO activity and catecholamine analysis The activity of MAO-A and MAO-B was determined by the method adapted from Tipton et al. (1982). Briefly, protein homogenate was added to a suitable dilution of the enzyme preparation. The mixture was incubated with 0.15 mM L-deprenyl or clorgyline (for determination of MAO-A or MAO-B, respectively). Incubation  was carried for 1 hour at 37 C prior the addition of the radioactive 14 metabolites, C-5-hydroxytryptamine binoxalate (100 mM, 30 minutes) for determination of MAO-A or 14C-phenylethylamine (100 mM, 20 minutes) for determination of MAO-B. The reaction was stopped with ice-cold citric acid (2 M), and the radioactivity determined by liquid-scintillation counter. The content of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA); 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and noradrenalin (NE) was determined in the striatum and hippocampus by high performance liquid chromatography system, as previously described (Gal et al., 2005). The levels were calculated by comparison with monoamine standards (Sigma, St. Louis, MO, USA) in known concentrations and normalized relative to tissue weight. 2.8. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction analyses For Western blotting analyses, hippocampal and striatal samples were homogenized in Tris-sucrose buffer pH 7.4 (containing a mixture of protease inhibitors, Roche, Inc and phosphatase

inhibitors), separated by SDS-PAGE (4%e12% Bis-Tris gels) and blotted on Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), as previously described (Kupershmidt et al., 2012b). Protein content was determined using the Bradford method. Detection was achieved using Western blotting detection reagent, ECL system (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK). Quantitation of the labeled bands in the autoradiograms was accomplished by measuring the optical density, using the computerized imaging program Bio-1D (Vilber Lourmat Biotechnology, Marne de Vallee, France). For the determination of gene expression, RNA was isolated from hippocampal samples using a PurfectPure RNA Cultured Cell Kit (50 PRIME Inc, MD, USA), as recommended by the manufacturer and reverse transcribed, as previously described (Kupershmidt et al., 2011). Real-time RT-PCR was performed with specific primers, for the genes in search (including, tyrosin kinase B [TrkB], glial cellderived neurotrophic factor [GDNF], nerve growth factor [NGF], Bax, and Bcl-2, purchased from Qiagen, Germany) on the provided program of the LightCycler system (Roche Applied Science). The relative expression level of a given mRNA was assessed by normalizing to the housekeeping genes 18S rRNA, g-tubulin and/or b-actin, compared with control values. 2.9. Immunohistochemistry analysis Brain hemispheres were post fixed in 4% (vol/0.1 M PBS vol) paraformaldehyde (48 hours, 4  C) and cryoprotected by 30% sucrose (48 hours, 4  C). Six series of 40-mm coronal sections of the hippocampus were collected in PBS on a freezing-sliding microtome. To identify viable and nonviable stained cells in the dentate gyrus (DG) and CA3 regions, the sections were stained with cresyl violet (Nissl staining). For immunohistochemistry studies, staining slides were incubated in PBS containing 10% donkey serum at 37  C for 1 hour to block nonspecific staining. Next, slides were rinsed 3 times in PBS, and coverslips were mounted with Vectashield containing 40 -6diamidino-2-phenylindole for nuclear staining. Immunofluorescence was performed, using the antibodies against BDNF and pCREB

Table 1 Effect of VAR on striatal levels of amines and their metabolites Group

DA Control VAR (1.1 mg/kg) VAR (3.3 mg/kg) VAR (10 mg/kg)

(DOPAC þ HVA)/DA

Striatal levels (pmol/mg)

39.1 57.8 54.7 48.2

DOPAC    

3.3 11 8.2 6.7

2.7 2.7 2.8 1.4

   

0.2 0.9 0.2 0.4a

HVA 5.7 5.1 5.9 3.2

   

Striatal levels (pmol/mg) 5-HT

0.4 1.4 0.4 0.6a

0.22 0.20 0.17 0.095

   

0.016 0.015 0.024 0.011a

8.2 10.5 10.9 12.6

   

5-HIAA 0.7 0.8 0.8a 0.8a

2.8 3.0 2.9 2.1

   

0.1 0.3 0.2 0.3a

5-HIAA/5-HT NE 1.2 2.1 1.6 1.7

   

0.08 0.3a 0.2 0.1a

0.35 0.29 0.27 0.16

   

0.028 0.041 0.018a 0.019a

Mice received either vehicle (control) or VAR (1.1, 3.3, or 10 mg/kg, p.o.) for 14 consecutive days. Striatal levels of amines and their metabolites were determined by HPLC analysis. DA and 5-HT metabolism was expressed as the ratio (DOPAC þ HVA)/DA and 5-HIAA/5-HT, respectively. Results represent mean  SEM (n ¼ 7). Key: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; NE, noradrenalin; p.o., per os; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus control.

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Fig. 3. Effects of VAR treatment on MAO activity in rat brain and peripheral tissues and on rat BP response to tyramine. Rats were treated with vehicle (saline; control) or increasing doses of VAR 16 hours before killing. (A) MAO-A and MAO-B activities were determined in brain, liver, and small intestine. Results are expressed as percentage inhibition of control and represent the mean  SEM (n ¼ 7e8); *p < 0.05 versus respective controls. (B) BP potentiation in response to increasing doses of tyramine after acute treatment with saline, VAR, or TCP. Results are expressed as mean arterial BP (MABP) (mmHg) and represent the mean  SEM (n ¼ 5e7); *p < 0.05 versus vehicle-treated rats. #p < 0.05 versus TCP-treated rats. Abbreviations: MABP, mean arterial blood pressure; MAO, monoamine oxidase; TCP, tranylcypromine; SEM, standard error of the mean; VAR, VAR10303.

by reaction with N-propargylamine (compound 1.5), which yields the target compound VAR (compound 1.6). The composition of the iron complex of VAR was established by spectrophotometric measurements. Fig. 1B shows that under the experimental conditions, a complex of 1:3 (iron:VAR) molar ratio was formed. Spectrophotometric studies reveals the complex formation of VAR with Fe(III), Cu(II), and Zn(II) but with higher selectivity for Fe(III) over the last 2 metal ions (data not shown). The iron-chelating potency of VAR was determined and compared with that of the prototype iron chelator deferoxamine (DFO), by assessing the ability of the 2 compounds to compete with ferrozine for Fe(II) ions (Fig. 1C). IC50 value was estimated as the drug concentration required for 50% inhibition of the iron-ferrozine complex and was calculated as: DFO ¼ 7.81 mM and VAR ¼ 44.2 mM (Fig. 1C). Compared with DFO, the lower potency of VAR in competing for iron is beneficial, because the high affinity of DFO for iron may restrict its use for prolonged periods of time in iron-overload unrelated conditions, as a result of its serious cytotoxicity effects (Zheng et al.,

and analyzed in Bio Rad Radiance 2000 confocal system, supported with Laser-Sharp 2000 software. Eight images of slides were obtained per each region of interest, and data analysis was calculated for separate images, taken for each rat and expressed as the mean  standard error of the mean of 3 animals per group. 2.10. Statistical analysis Differences among means were analyzed using 1-way analysis of variance, and results were expressed as the means  standard error of the mean; p-values < 0.05 were considered significant. 3. Results 3.1. Chemical synthesis, iron-chelating, and antioxidant properties of VAR Fig. 1A shows the synthesis of VAR, starting from commercially available 8-hydroxyquinoline (compound 1.1) and ending Table 2 Effect of a single VAR administration on striatal levels of amines and their metabolites Group

DA Control VAR (7.5 mg/kg) VAR (10 mg/kg) VAR (12.5 mg/kg)

(DOPAC þ HVA)/DA

Striatal levels (pmol/mg)

38.7 47.0 52.2 39.8

DOPAC    

2.5 2.1a 4.4a 2.5

4.6 2.7 2.1 1.4

   

0.75 0.16a 0.41a 0.16a

HVA 2.70 1.70 1.67 1.50

Striatal levels (pmol/mg) 5-HT

   

0.40 0.07a 0.20a 0.36

0.18 0.09 0.074 0.064

   

0.024 0.002a 0.008a 0.004a

3.3 4.9 4.4 4.2

   

5-HIAA 0.24 0.33a 0.27a 0.32

1.83 1.73 1.54 1.42

   

0.15 0.07 0.13 0.17

5-HIAA/5-HT NE 0.48 0.72 0.83 0.92

   

0.03 0.14 0.07a 0.07a

0.56 0.35 0.33 0.35

   

0.04 0.02a 0.05a 0.07a

Rats received either saline (control) or VAR (7.5, 10, and 12.5 mg/kg s.c.) 16 hours before killing. The contents of striatal amine and their metabolites were determined by HPLC analysis. DA and 5-HT metabolism was expressed as the ratio (DOPAC þ HVA)/DA and 5-HIAA/5-HT, respectively. Results represent mean  SEM (n ¼ 5). Key: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; NE, noradrenalin; s.c., subcutaneous; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus control.

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Fig. 4. Neuroprotective effect of VAR on apomorphine-induced rotational behavior and on DA and 5-HT turnover in 6-OHDA-induced striatal lesion. (A) Apomorphine-induced rotation test. The results are expressed as ipsilateral net turns/60 minutes. (B) Striatal DA turnover, (C) 5-HT turnover, and (D) hippocampal 5-HT turnover were determined by HPLC analysis. DA and 5-HT metabolism was expressed as the ratio (DOPAC þ HVA)/DA and 5-HIAA/5-HT, respectively. Results represent the mean  SEM (n ¼ 5). #p < 0.05 versus control (sham), *p < 0.05 versus 6-OHDA-treated rats. Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HT, serotonin; 5-HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; 6-OHDA, 6-hydroxydopamine; SEM, standard error of the mean; VAR, VAR10303.

2005a). In addition, VAR showed high potency in inhibiting lipid-peroxidation, as measured by the formation of malondialdehyde (MDA), with IC50 of 1.47  0.01 mM (n ¼ 3); (p < 0.05) (Fig. 1D). 3.2. Effects of VAR on MAO-A and MAO-B inhibition; striatal levels of amines and metabolites To analyze the effect of systemic VAR treatment on MAO-A and MAO-B inhibition in brain and peripheral tissues and on striatal levels of amines and metabolites, mice were treated with vehicle or increasing drug concentrations (1.1, 3.3, and 10 mg/kg, p.o., daily for 14 days). At the end of VAR treatment, liver iron levels were not significantly different between vehicle- and VAR-treated mice (7.5  0.74 and 7.04  0.18 mg iron/100 mg tissue, respectively, p > 0.05).

Fig. 2 shows that VAR administration resulted in a significant dose-dependent MAO-A and MAO-B inhibition in the brain. VAR (10 mg/kg) administration induced a marked inhibition of both MAO-A and MAO-B in the brain, with no inhibition of MAO-A in the liver and intestine, and only little inhibition of MAO-B in the intestine (Fig. 2). In accordance, VAR administration increased striatal DA and 5-HT levels (Table 1). VAR at 10 mg/kg significantly reduced striatal levels of DOPAC and HVA, as well as of 5-HIAA, as compared with controls, and this is reflected in the decreased turnover of striatal DA and 5-HT (Table 1). 3.3. Limited potentiation of BP in response to tyramine by VAR To assess the BP potentiation in response to tyramine, rats received either vehicle (saline) or various doses of VAR, and the

Table 3 Neuroprotective effect of VAR on striatal levels of amines and their metabolites in 6-OHDA-lesioned rats Group

Striatal levels (pmol/mg) DA

DOPAC

HVA

5-HT

5-HIAA

NE

Sham 6-OHDA 6-OHDA þ VAR (10 mg/kg)

39.5  3.5 11.5  1.9a 19.3  3.4b

5.4  0.4 2.2  0.3a 0.4  0.09b

2.8  0.3 1.4  0.1a 0.3  0.04b

4.0  0.2 4.1  0.2 9.1  0.6b

1.8  0.07 2.3  0.1a 0.9  0.08b

1.05  0.2 0.6  0.1 1.8  0.4b

Levels of amines and their metabolites in the striatum were determined by HPLC analysis. Results represent mean  SEM (n ¼ 5). Key: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; NE, noradrenalin; 6-OHDA, 6-hydroxydopamine; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus sham. b p < 0.05 versus 6-OHDA-lisioned rats.

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Table 4 Neuroprotective effect of VAR on hippocampal levels of 5-HT, 5-HIAA and NE in 6-OHDA-lesioned rats Group

Hippocampal levels (pmol/mg) 5-HT

5-HIAA

NE

Sham 6-OHDA 6-OHDA þ VAR (10 mg/kg)

2.3  0.4 1.9  0.1 4.4  0.4b

2.4  0.09 2.8  0.2 1.1  0.1b

3.7  0.2 3.1  0.1a 3.7  0.4

Levels of 5-HT, 5-HIAA and NE in the hippocampus were determined by HPLC analysis. Results represent mean  SEM (n ¼ 5). Key: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HPLC, high performance liquid chromatography system; NE, noradrenalin; 6-OHDA, 6-hydroxydopamine; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus sham. b p < 0.05 versus 6-OHDA-lisioned rats.

effects of the drug on MAO inhibition and mean arterial BP (MABP) were assessed. The inhibition of MAO-A and MAO-B in brain, liver, and small intestine of rats following VAR administration is shown in Fig. 3. Acute VAR administration (7.5, 10, or 12.5 mg/kg, s.c.) resulted in a significant dose-dependent MAO-A and MAO-B inhibition in the brain, up to approximately 70% and 50% inhibition of MAO-A and MAO-B, respectively (Fig. 3A). However, very low MAO-A and MAO-B inhibition was noted in liver and small intestine in all drug dosages used (Fig. 3A). Fig. 3B shows that in saline-treated rats, up to 50 mg/kg oral tyramine did not significantly increase MABP, whereas in TCP-treated rats, only 10 mg/kg of tyramine was required to increase MABP by approximately 30 mmHg. Similar to vehicle (saline)-treated animals, at all 3 VAR dosages used, up to approximately 25 mg/kg of tyramine did not increase MABP by approximately 30 mmHg. At 12.5 mg/kg of VAR, a dose of 50 mg/kg tyramine was needed to increase MABP by levels higher than 30 mmHg (Fig. 3B). These findings indicate a limited potentiation of BP response to tyramine by VAR. The effects of acute VAR treatment on the levels of striatal amines and metabolites are shown in Table 2. VAR (7.5 and 10 mg/ kg) treatment significantly increased the striatal levels of DA and 5HT and reduced DOPAC and HVA. The turnover of both amines, as expressed by the ratios DOPAC þ HVA/DA and 5-HIAA/5-HT, was markedly reduced, as compared with controls (Table 2). 3.4. Animal models of PD We further investigated the neuroprotective and neurorescue effects of VAR against striatal dopaminergic neurodegeneration induced by 6-OHDA, as well as MPTP lesions in rats and mice, respectively, as it is important to use different animal models and species to evaluate a drug’s therapeutic potency. 3.4.1. Neuroprotective effect of VAR in 6-OHDA-lesioned rats The unilateral 6-OHDA lesion model of PD has the advantage of easily assessing the side biased motor impairments by utilizing

Fig. 5. Neurorescue effect of VAR on striatal DA turnover and TH protein levels in MPTP-treated mice. (A) Striatal DA metabolism was expressed as the ratio (DOPAC þ HVA)/DA. (B) Striatal TH levels were assessed by Western blotting analysis. The loading of the lanes was normalized to levels of b-actin. Results represent the mean  SEM (n ¼ 6e9). #p < 0.05 versus control, *p < 0.05 versus vehicle-treated MPTP mice. Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SEM, standard error of the mean; TH, tyrosine hydroxylase; VAR, VAR10303.

drug-induced rotation test (Ungerstedt and Arbuthnott, 1970). As DA deficiency leads to the super-sensitivity of striatal DA receptor, the non-selective DA receptor agonist, apomorphine is commonly used to determine the degree of dopaminergic degeneration (Deumens et al., 2002). Fig. 4A shows that apomorphine-induced asymmetrical rotational behavior was significantly reduced in 6OHDA/VAR-treated rats, compared with 6-OHDA/vehicle-treated rats. Intrastriatal 6-OHDA lesion induced a marked depletion of

Table 5 Neurorescue effect of VAR on striatal levels of amines and their metabolites in MPTP-treated mice Group

Striatal levels (pmol/mg) DA

DOPAC

HVA

5-HT

5-HIAA

NE

Control MPTP MPTP þ VAR (20 mg/kg)

47.8  4.9 13.5  1.7a 27.4  5.7b

5.6  0.5 2.5  0.3a 3.0  0.5b

4.2  0.2 2.7  0.2a 3.2  0.4b

2.3  0.2 2.4  0.2 3.3  0.3b

1.7  0.1 1.9  0.1 2  0.2

0.57  0.1 0.67  0.1 1.23  0.4

Levels of amines and their metabolites in the striatum were determined by HPLC analysis. Results represent mean  SEM (n ¼ 6e9). Key: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NE, noradrenalin; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus control. b p < 0.05 versus MPTP-treated mice.

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Table 6 Neurorescue effect of VAR on hippocampal levels of 5-HT, 5-HIAA and NE in MPTPtreated mice Group

Hippocampal levels (pmol/mg) 5-HT

5-HIAA

NE

Control MPTP MPTP þ VAR (20 mg/kg)

2.2  0.2 2.1  0.3 3.1  0.3a

1.7  0.2 1.9  0.2 2.0  0.3

1.8  0.03 1.7  0.1 1.9  0.1

Levels of 5-HT, 5-HIAA and NE in the hippocampus were determined by HPLC analysis. Results represent mean  SEM (n ¼ 6e9). Key: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HPLC, high performance liquid chromatography system; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; NE, noradrenalin; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus MPTP-treated mice.

striatal DA and its metabolites, DOPAC and HVA (Table 3), resulting in a significant increase in DA turnover in the lesioned striata, compared with the sham control group (Fig. 4B). VAR (10 mg/kg) treatment significantly attenuated the loss of striatal DA and markedly reduced DA turnover, compared with the vehicle-treated 6-OHDA group (Table 3 and Fig. 4B). In addition, the levels of 5-HT were significantly elevated (Table 4) and turnover of 5-HT was reduced (Fig. 4C and D) in the striatum and hippocampus of 6OHDA/VAR-treated rats, compared with 6-OHDA/vehicle-treated rats. The in vitro neuroprotective capacity of VAR against oxidative stress was assayed by using human neuroblastoma SH-SY5Y cells and 2 neurotoxic models; H2O2, for the generation of exogenous free radicals and 6-OHDA, as a selective dopaminergic neurotoxin. The data showed that incubation of SH-SY5Y cells with VAR (10 mM) elicited a significant protective effect against both H2O2- and 6-OHDA-induced neurotoxicity (Supplementary Material and Supplementary Fig. 1).

3.4.2. Neurorescue effect of VAR in MPTP mice Neurorestorative effects of VAR were tested using the MPTPinduced mouse model of PD, as described in Section 2. MPTP administration to mice resulted in a significant decrease in striatal DA levels, as well as in DA metabolites, DOPAC, and HVA versus control mice (Table 5). In the neurorescue paradigm, a continuous administration of VAR, given following MPTP, significantly prevented the decline in striatal DA levels, compared with MPTP/ vehicle-treated mice (Table 5), and fully restored the increased striatal DA turnover, induced by MPTP (Fig. 5A). In addition, 5-HT levels were increased in the striatum (Table 5) and hippocampus (Table 6) of MPTP/VAR-treated, compared with MPTP/vehicletreated mice, which led to decreased turnover of 5-HT in both brain regions. Western blotting analysis showed that MPTP markedly reduced striatal TH levels, whereas VAR treatment significantly increased TH levels of MPTP/VAR-treated versus MPTP/vehicletreated mice (Fig. 5B). 3.4.3. Effect of VAR in aged rats To evaluate the possible neuroprotective effects of VAR on agerelated cognitive deficits, 18-month-old rats were chronically treated with VAR and subjected to several behavioral tests. The open field exploration behavioral test demonstrated a significant induction of locomotor activity in VAR-treated aged group, as indicated by a higher number of crossings and rearings and a reduced latency to start locomotion, compared with vehicletreated aged rats (Fig. 6A). In the object recognition test, VARtreated animals exhibited a significantly higher preference in exploring the novel object (higher recognition index) during shortterm memory and long-term memory retention trials than vehicletreated aged rats (Fig. 6B). Results for the forced swim test, known as a behavioral despair test (Pirondi et al., 2005) that is commonly used to test for assessment of depression in animal models (Borsini and Meli, 1988), demonstrated a significant reduction of time spent

Fig. 6. Effects of VAR on age-related behavioral impairments. Aged rats were treated with vehicle (saline) or VAR (1 mg/kg; s.c., 4 times weekly for 6 months) and subjected to several behavioral tests, namely: (A) open field exploration behavior test; (B) object recognition test (short-term memory [STM], 1.5 hours after training and long-term memory (LTM), 24 hours after training); and (C) forced swim test. Each bar represents the mean  SEM (n ¼ 7e8); # p < 0.05 versus vehicle-treated young rats; *p < 0.05 versus vehicletreated aged rats. Abbreviations: s.c., subcutaneous; SEM, standard error of the mean; VAR, VAR10303.

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Table 7 Effect of VAR on striatal levels of amines and their metabolites in aged rats Treatment

Striatal levels (pmol/mg) DA

DOPAC

HVA

5-HT

5-HIAA

NE

Young Aged Agedþ VAR (1 mg/kg)

43.2  10.1 35.1  5.9a 34.7  2.8

2.02  0.15 1.7  0.16a 0.99  0.05b

1.09  0.25 1.19  0.22 0.58  0.05b

1.31  0.32 0.94  0.21a 1.39  0.33b

1.53  0.31 1.24  0.37 1.20  0.33

0.81  0.14 0.41  0.18a 0.51  0.23

Levels of amines and their metabolites in the striatum were determined by HPLC analysis and expressed as the mean  SEM (n¼ 7e8). Key: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; HPLC, high performance liquid chromatography system; NE, noradrenalin; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus vehicle-treated young rats. b p < 0.05 versus vehicle-treated aged rats.

in immobility in the VAR-treated aged group, compared with vehicle-treated aged rats (Fig. 6C). After the behavioral assessment, we examined the effect of VAR on MAO inhibition and levels of amines and their metabolites in the striatum and hippocampus of aged rats. Determination of cerebral MAO-A and MAO-B activities in response to VAR treatment showed that in VAR-treated aged rats, the enzymes were significantly inhibited by 55.5%  9.7% and 43.9%  13.4% of control, respectively (p < 0.05). In addition, a significant reduction was observed in the levels of striatal DA metabolites, DOPAC, and HVA of VAR-treated rats, compared with vehicle-treated aged rats (Table 7). In the hippocampus, VAR significantly increased the levels of 5-HT and NE and decreased the levels of 5-HT metabolite, 5-HIAA, compared with vehicle-treated aged rats (Table 8). The regulatory effect of VAR on expression levels of Bcl-2, Bax, and various growth factors was further determined in the hippocampus of aged rats. We have shown that VAR administration to aged rats significantly increased hippocampal Bcl-2 mRNA expression (Fig. 7A) and protein levels (Fig. 7C) and decreased Bax mRNA expression (Fig. 7B) and protein levels (Fig. 7D), compared with vehicle-treated aged rats. The ratio of Bcl-2/Bax, which correlates with cellular apoptosis, was significantly increased (approximately 7 folds) in VAR-treated rats, compared with vehicle-treated aged rats. Immunostaining and Western blotting analyses demonstrated that hippocampal BDNF levels were significantly increased in VARtreated versus vehicle-treated aged rats (Fig. 8A and B). VAR also upregulated the hippocampal mRNA expression levels of BDNF receptor, TrkB (by 1.72  0.2-folds, p < 0.05), GDNF (Fig. 8C) and NGF (Fig. 8D), compared with vehicle-treated aged rats. Analysis of signaling pathway cascades, implicated in neuronal survival and synaptic plasticity molecular processes reveals that VAR treatment significantly increased the levels of hippocampal pERK1/2 (Fig. 9A) and pCREB (Fig. 9B and C) in drug-treated versus vehicle-treated aged rats. Additionally, VAR treatment in aged rats significantly increased hippocampal protein levels of the neuronal specific markers, GAP-43 (Fig. 10A) and synaptophysin (Fig. 10B). Table 8 Effect of VAR on hippocampal levels of 5-HT, 5-HIAA and NE in aged rats Treatment

Hippocampal levels (pmol/mg) 5-HT

5-HIAA

NE

Young Aged Aged þ VAR (1 mg/kg)

2.02  0.22 2.08  0.11 2.89  0.24b

0.84  0.08 1.22  0.19a 0.95  0.12b

0.33  0.17 0.19  0.09a 0.37  0.24b

Levels of 5-HT, 5-HIAA and NE in the hippocampus were determined by HPLC analysis and expressed as the mean  SEM (n¼ 7e8). Key: 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HPLC, high performance liquid chromatography system; NE, noradrenalin; SEM, standard error of the mean; VAR, VAR10303. a p < 0.05 versus vehicle-treated young rats. b p < 0.05 versus vehicle-treated aged rats.

Hippocampal mRNA expression levels of synapsin-1 were also upregulated by 1.64  0.2-folds (p < 0.05) in VAR-treated versus vehicle-treated aged rats. In support, we have demonstrated that VAR has the ability to induce neurite outgrowth and increase the levels of GAP-43 and synaptophysin in SH-SY5Y cells, compared with vehicle-treated cells (Supplementary Materials and Supplementary Fig. 2). 4. Discussion In searching for antioxidant/iron chelators with MAO inhibitory and neuroprotective properties, we have designed and synthesized a series of multifunctional brain permeable compounds for agerelated neurodegenerative diseases (Zheng et al., 2005a). Here, we show that among these novel compounds, VAR, which amalgamates the propargyl moiety of rasagiline with the backbone of the iron chelator VK28, affords iron chelation, iron-induced lipidperoxidation inhibitory potency and brain selective MAO-A and MAO-B inhibitory effects. In addition, VAR was found to exert neuroprotective and/or neurorescue activity in rat 6-OHDA- and mouse MPTP- PD models and beneficial effects on age-related alterations in rats. VAR, being N-propargylamine derivative, caused a selective brain inhibition of both MAO-A and MAO-B and in accordance, significantly reduced the levels of the DA metabolites, DOPAC, and HVA in the striatum. Also, the increases in the MAO-A substrate, 5HT in the striatum and hippocampus correlated brain MAO-A inhibitory effect of VAR. These properties of inhibition of brain MAO-A and MAO-B activity and induction of 5-HT levels suggest that VAR may have therapeutic and antidepressive effects in aging and neurodegenerative diseases. Indeed, depression and cognitive decline have been reported to be associated with aging and agerelated neurodegenerative diseases (McKinney et al., 2012), further emphasizing the need of multifunctional drugs. An important finding is the little inhibition of MAO-A and MAO-B in the liver and small intestine following VAR administration, as irreversible high MAO-A inhibition in the periphery is associated with potentiation of tyramine-induced cardiovascular activity (“cheese effect”) (Finberg and Tenne, 1982; Finberg and Youdim, 1988). The present data show that VAR produced only limited potentiation of blood pressure in response to oral tyramine, making the drug potentially useful for the treatment of patients with PD and depressive illness. This is consistent with previous studies with the multifunctional M30 demonstrated that the compound selectively inhibited brain MAO-A and MAO-B and had a limited pressor effect (Gal et al., 2010b). In the present study, the neuroprotective effect of VAR was first detected in the 6-OHDA-rat model, one of the classical PD animal models (Zigmond and Stricker, 1984). VAR treatment attenuated apomorphine-induced rotational behavior and prevented the reduction in striatal DA content and the increase in DA turnover,

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Fig. 7. Effect of VAR on the expression of hippocampal Bcl-2 and Bax in aged rats. Aged rats were treated as described in Fig. 6. mRNA expression levels of (A) Bcl-2 and (B) Bax were assessed by quantitative real-time RT-PCR. The amount of each product was normalized to the geometric mean of 3 housekeeping genes: 18S-rRNA, b-actin, and g-tubulin. Representative Western blotting bands and quantitative analysis of (C) Bcl-2 and (D) Bax protein levels. Values are normalized to the levels of b-actin and are the mean  SEM (n ¼ 7e8); *p < 0.05 versus control (vehicle-treated aged rats). Abbreviations: mRNA, messenger RNA; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; VAR, VAR10303.

normally observed in 6-OHDA-lesioned rats (Blandini et al., 2004; Mendez and Finn, 1975). Further support for the neuroprotective effects of VAR has come from our studies, evaluating the possible neurorescue activity of the drug in the post-MPTP-induced nigrostriatal DA neurodegenerative model in mice. In this neurorescue paradigm, we showed that administration of VAR managed to restore striatal DA turnover and increase striatal DA levels and TH protein expression. These neuroprotective effects might be related to the multimodal design strategy of VAR, possessing both propargyl and antioxidant/iron chelating moieties. Given that products of the MAO-catalyzed reaction (e.g., aldehydes and hydrogen peroxide) are compelling inducers of lipid peroxidation, it is assumed that inhibition of MAO in the brain is associated with neuroprotective effects in neurodegenerative and age-related disturbance of the homeostasis and generation of free radicals in involution of the nervous system (Kumar and Andersen, 2004; Shemyakov, 2001). Thus, studies in various PD models have demonstrated that propargyl-containing derivatives (e.g., rasagiline, ladostigil, and M30) possess neuroprotective activity; this was attributed to inhibition of MAO, as well as induction of anti-apoptotic Bcl-2 family protein (Bcl-2, Bcl-W, and Bcl-xL) and the protein kinase C isoforms a and ε, upregulation of BDNF and GDNF and activation of RAS-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT (Weinreb et al., 2010, 2012; Youdim et al., 2014). Additionally,

neuroprotection can be associated with antioxidant/iron chelating properties of VAR, as there is increasing evidence that iron accumulates in its reactive from in the brain during aging and PD and is involved in neurodegenerative processes (Jellinger, 1999; Jellinger et al., 1993; Zecca et al., 2004). It was shown that iron levels were increased in the substantia nigra pars compacta of 6-OHDA- and MPTP-lesioned animals (rats, mice, and monkeys) (Gerlach et al., 2000; He et al., 1996; Youdim et al., 2004; Zecca et al., 2004). In fact, previous studies have demonstrated neuroprotective activity with genetic or pharmacologic iron chelators (DFO, VK28, and M30) in various PD neurotoxic models, such as MPTP, 6-OHDA, and lacatcystin (Ben-Shachar et al., 1991, 2004; Gal et al., 2005, 2010b; Zhu et al., 2007). Indeed, VK28 and M30 attenuated lacatcystininduced iron accumulation in the ventral midbrain in mice (Zhu et al., 2007). In addition, M30 was recently shown to significantly reduce cerebral iron accumulation in aged (Kupershmidt et al., 2012a), APP/PS1 transgenic Alzheimer’s disease (Kupershmidt et al., 2012b) and SOD1-G93A transgenic Amyotrophic lateral sclerosis (Wang et al., 2011) mice. The in vivo effect of VAR on neuronal iron accumulation remains to be confirmed in future experiments. Finally, we demonstrated in the current work that VAR can induce neuroprotective effects on age-related changes in neurobehavioral functions. Here, we report that systemic chronic treatment with VAR in aged rats exerted a significant beneficial

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Fig. 8. Effect of VAR on the expression of hippocampal neurotrophic factors in aged rats. (A) Representative images of hippocampal DG and CA3 regions stained for BDNF, Nissl, and DAPI of control and VAR-treated aged rats. Data present the mean fluorescent intensity (MFI)  SEM. (B) Representative Western blot image and quantitative analysis of hippocampal BDNF levels. Values are normalized to levels of b-actin and are expressed as the mean  SEM (n ¼ 5). mRNA expression levels of (C) GDNF and (D) NGF, assessed by quantitative real-time RT-PCR. The amount of each product was normalized to the geometric mean of 3 house-keeping genes: 18S-rRNA, b-actin, and g-tubulin. Data are expressed as arbitrary units of gene expression and represent the mean  SEM (n ¼ 7e8). *p < 0.05 versus control (vehicle-treated aged rats). Abbreviations: BDNF, brain-derived neurotrophic factor; DAPI, 40 -6-diamidino-2-phenylindole; DG, dentate gyrus; GDNF, glial cell-derived neurotrophic factor; NGF, nerve growth factor; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; VAR, VAR10303. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

effect on neuropsychiatry functions and cognitive behavior deficits, assessed by open field, novel object recognition, and forced swim tests. In this context, positive impact on cognitive agerelated impairment was recently observed following chronic treatment of aged mice with M30 (Kupershmidt et al., 2012a). In fact, aging is well characterized by a progressive decline of cognitive performance, mainly attributed to structural and functional alterations of the hippocampus (Elgersma and Silva, 1999). Especially, neurotropic factors have been considered as essential molecular mediators involved in survival, outgrowth, and differentiation of neurons (Poo, 2001). Thus, we further assessed the impact of VAR on neuroprotective-associated molecular targets in the hippocampus of aged rats, and provided the following observation: (1) chronic VAR treatment significantly enhanced the hippocampal levels of BDNF in the DG and CA3, and elevated the expression levels of TrkB and synaptic plasticity markers, including synapsin-I (associates with the structural and functional organization of the presynaptic terminals and synaptic vesicles (Greengard et al., 1993); GAP-43 (an intrinsic determinant in the establishment and reorganization of synaptic connections) and synaptophysin (one of the main integral proteins of synaptic vesicles). In accordance, we showed that VAR can stimulate neurite outgrowth and upregulate expression levels of GAP-43 and

synaptophysin in SH-SY5Y cells; (2) the expression levels of GDNF and NGF mRNAs were upregulated, further indicating that the beneficial response of the drug might be attributed, at least in part, to its effect on hippocampal synaptic growth and/or function; (3) our results also suggest that VAR could modulate hippocampal levels of various molecular systems that are involved with the signaling of growth factors, such as pERK 1/2 and pCREB (Impey et al., 2004; Li et al., 2009). These signaling pathways are stimulated by the interaction of growth factors with their respective receptors and play a critical role in the regulation of neuronal survival (Schindowski et al., 2008), so they could partially be involved in neuroprotective and neurorestorative processes. Another interesting finding of this study is the upregulation of mRNA and protein levels of the prosurvival Bcl-2 and reduction of the proapoptotic Bax, which is in keeping with previous studies demonstrating prosurvival effects of propargyl-containing derivatives that are mediated by the regulation of the Bcl-2 family proteins (Akao et al., 2002; Bar-Am et al., 2005). In summary, chelation therapy was previously introduced as a novel therapy concept and rational for the development of metalbinding drugs for age-related neurodegeneration. Hence, there are only a small number of iron chelating compounds that are under research and some are in their early stage of development and

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Fig. 9. Effect of VAR on hippocampal levels of pERK1/2 and pCREB in aged rats. (A) pERK1/2 and (B) pCREB were evaluated by immunoblotting. Values are normalized to the levels of b-actin. (C) pCREB and Nissl immunostaining were examined in the hippocampal DG region of vehicle- and VAR-treated aged rats. Data present the MFI  SEM. Values are expressed as the mean  SEM. *p < 0.05 versus vehicle-treated aged rats. Abbreviations: CREB, cAMP response element-binding protein; DG, dentate gyrus; ERK, extracellular signal-regulated kinase; MFI, mean fluorescent intensity; SEM, standard error of the mean; VAR, VAR10303. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

thus, further research as that one, are vital to endorse this newly therapeutic strategy. Moreover, because of the complex nature of the age-related neurodegenerative disorders, it is important that pharmacologic intervention for these diseases would act through multiple pathological pathways. Indeed, this study demonstrates

that the novel multimodal iron chelator, VAR exerted neuroprotective and neurorescue effects in PD models and aged animals, further suggesting that a drug that can affect multiple brain targets could provide an ideal treatment for age-associated neurodegenerative diseases.

Fig. 10. Effect of VAR on hippocampal levels of GAP-43 and synaptophysin in aged rats. Representative Western blotting image and quantitative analysis of hippocampal levels of (A) GAP-43 and (B) synaptophysin levels in aged rats. Values represent the mean  SEM (n ¼ 7e8); *p < 0.05 versus vehicle-treated aged rats. Abbreviations: GAP-43, growth-associated protein; SEM, standard error of the mean; VAR, VAR10303.

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