The 5α-reductase inhibitor Dutasteride but not Finasteride protects dopamine neurons in the MPTP mouse model of Parkinson's disease

Neuropharmacology 97 (2015) 86e94

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The 5a-reductase inhibitor Dutasteride but not Finasteride protects dopamine neurons in the MPTP mouse model of Parkinson's disease
lanie Bourque a, b, Sara Al Sweidi a, b, Marc Morissette a, Nadhir Litim a, b, Me
re se Di Paolo a, b, * The a b

Neuroscience Research Unit, Centre Hospitalier Universitaire de Qu
ebec, CHUL, Quebec City, Canada Faculty of Pharmacy, Laval University, Quebec City, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 January 2015 Received in revised form 17 April 2015 Accepted 11 May 2015 Available online 23 May 2015

Finasteride and Dutasteride are 5a-reductase inhibitors used in the clinic to treat endocrine conditions and were recently found to modulate brain dopamine (DA) neurotransmission and motor behavior. We investigated if Finasteride and Dutasteride have a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) male mice as a model of Parkinson's disease (PD). Experimental groups included saline treated controls and mice treated with saline, Finasteride (5 and 12.5 mg/kg) or Dutasteride (5 and 12.5 mg/kg) for 5 days before and 5 days after MPTP administration (4 MPTP injections, 6.5 mg/kg on day 5 inducing a moderate DA depletion) and then they were euthanized. MPTP administration decreased striatal DA contents measured by HPLC while serotonin contents remained unchanged. MPTP mice treated with Dutasteride 5 and 12.5 mg/kg had higher striatal DA and metabolites (DOPAC and HVA) contents with a decrease of metabolites/DA ratios compared to salinetreated MPTP mice. Finasteride had no protective effect on striatal DA contents. Tyrosine hydroxylase (TH) mRNA levels measured by in situ hybridization in the substantia nigra pars compacta were unchanged. Dutasteride at 12.5 mg/kg reduced the effect of MPTP on specific binding to striatal DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) measured by autoradiography. MPTP reduced compared to controls plasma testosterone (T) and dihydrotestosterone (DHT) concentrations measured by liquid chromatographyetandem mass spectrometry; Dutasteride and Finasteride increased plasma T levels while DHT levels remained low. In summary, our results showed that a 5a-reductase inhibitor, Dutasteride has neuroprotective activity preventing in male mice the MPTP-induced loss of several dopaminergic markers. © 2015 Published by Elsevier Ltd.

Keywords: Finasteride Dutasteride MPTP Neuroprotection Dopamine Striatum

1. Introduction Parkinson's disease (PD), a slowly and progressive neurodegenerative disease, is caused by loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). This neuronal loss is accompanied with reduced striatal DA and its metabolites content and the rate-limiting enzyme of synthesis of DA tyrosine hydroxylase (TH) as well as a reduction of the dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) (Harrington et al., 1996). Clinical evolution of PD shows, in the early stage of

* Corresponding author. Neuroscience Research Unit, Centre Hospitalier Uni
bec, CHUL, 2705 Laurier Boulevard, Quebec City, Quebec G1V 4G2, versitaire de Que Canada. Tel.: þ1 418 654 2296; fax: þ1 418 654 2761. E-mail address: [email protected] (T. Di Paolo). http://dx.doi.org/10.1016/j.neuropharm.2015.05.015 0028-3908/© 2015 Published by Elsevier Ltd.

neurodegeneration, a loss of striatal terminals largely exceeding the loss of SNpc cell bodies (Burke and O'Malley, 2013). L-3,4dihydroxyphenylalanine (L-DOPA) or DA agonists are currently used to treat motor symptoms in patients with PD without effect on the DA neuronal loss or decline of motor function (Harrington et al., 1996; Shaw et al., 1980). Therefore, there is an urgent need to develop neuroprotective therapies to slow or halt progression of PD. During the last decade, a number of studies have focused on the effect of endogenous estrogen exposure or the use of hormone replacement therapy in PD (Smith and Dahodwala, 2014). Given that a higher incidence and earlier age of onset of PD in men were reported in many studies (Bourque et al., 2009), this suggests a beneficial effect of ovarian steroids on the development and progression of PD particularly in the early stages of the disease (Saunders-Pullman et al., 1999). A well-defined sex difference is

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also reported in the nigrostriatal tract physiology (Becker, 1999). Menopause, a state of decreased sex hormones levels, is associated with a deterioration in the condition of PD patients, whereas the risk of PD decreases with an increased duration of fertile lifespan (Ragonese et al., 2004). Hormone therapy has also been associated with a reduced risk of developing the disease (Currie et al., 2004). However, the beneficial effects of estrogen in the brain could be offset by adverse side effects; chronic use of steroid hormones has been associated with increased risk to develop breast and ovarian cancer (Calle et al., 2009). Furthermore, in men, elevated circulating estradiol levels may have unwanted side-effects such as enlarged breasts, weight gain, emotional disturbances as well as infertility (Lund et al., 1999; Rozati et al., 2002). Hence, this supports the search for drugs with estrogenic activity in the brain and avoiding the danger of elevated estrogens in men. A difference between males and females in the 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model in response to toxins is reported with a greater decrease in striatal DA content occurring in male compared to female mice (Miller et al., 1998). The ovarian steroids 17b-estradiol and progesterone (P) are reported to be protective to DA neurons against MPTP toxicity in male and female mice but not androgenic steroids such as testosterone (T) and dihydrotestosterone (DHT) (Morissette et al., 2008). Several studies reported that neuroactive steroids or neurosteroids such as P exhibit neuroprotective effects during neurodegenerative processes including PD (di Michele et al., 2013; Melcangi and Panzica, 2014). P is converted by the rate limiting enzyme 5a-reductase into dihydroprogesterone (DHP) (Russell and Wilson, 1994) then into allopregnanolone (AP) or isopregnanolone (Melcangi and Panzica, 2014). P (Callier et al., 2001) and AP (Adeosun et al., 2012) are reported to have neuroprotective effects in the MPTP mouse (Przedborski and Vila, 2006). Hence, considering the neuroprotective activity of several neurosteroids, modulation of their synthesis and/or metabolism could be an interesting strategy for potential therapies against neurode generative processes. Blockade of the 5a-reductase enzyme activity could be such an approach for the neuroprotection of nigrostriatal DA. Steroid 5a-reductases are a family of enzymes that catalyze the conversion of T into DHT and metabolize P. T is aromatized to 17b-estradiol (Finn et al., 2006). Finasteride and Dutasteride, inhibitors of 5a-reductase, are presently used in the treatment of conditions dependent on the metabolism of androgens such as benign prostatic hyperplasia and androgenic alopecia (Finn et al., 2006). In humans, Finasteride inhibits selectively 5a-reductase type II, and Dutasteride is reported to have higher potency than Finasteride in inhibiting both types I and II (100 and 3 times greater respectively) (Clark et al., 2004). Recently, Finasteride and Dutasteride have received attention regarding their important effects to modulate brain activities (Paba et al., 2011); preliminary clinical results highlight the 5a-reductase enzyme as a potential therapeutic target in the treatment of many diseases associated with dopaminergic hyperactivity (Paba et al., 2011). However, little is known on the effect of 5a-reductase inhibition in animal models of PD and its possible role in DA neuroprotection. In the light of these findings, we tested the hypothesis that Finasteride and Dutasteride could protect nigrostriatal DA against MPTP toxicity in male mice. We also hypothesized that these 5areductase inhibitors could prevent the decrease in T plasma levels induced by MPTP as previously observed (Bourque et al., 2014) that model the T reduction in human PD (Okun et al., 2002).

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2. Materials and methods 2.1. Animals Male C57BL/6 mice (10 weeks) were purchased from Charles River Canada (Montreal, QC, Canada). All animals were given food and water ad libitum and housed in cages (up to 3 per cage) at 22  C with a 12 h light/12 h dark cycle. Each experiment included 6e10 mice per treatment group. To avoid contamination, mice that received MPTP were housed separately from saline-treated control mice. The Laval University Animal Care Committee approved these animal studies. All efforts were made to minimize animal suffering and to reduce the number of mice used. 2.2. Drugs administration Mice were injected with Finasteride (Tocris, Ellisville, MO, USA) (5 or 12.5 mg/kg once daily (s.i.d.), intraperitoneal (i.p.)), Dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 or 12.5 mg/kg s.i.d., i.p.) or vehicle (0.9% saline with 1% tween 80 s.i.d., i.p.) for 10 days. On Day 5, mice received 4 injections of MPTP (Sigma Chemical, St. Louis, MO, USA) at 6.5 mg/kg (i.p.) at 2-h intervals (Fig. 1). Finasteride and Dutasteride were investigated in the present study at doses not affecting motor behavior. Indeed, Finasteride (60 or 100 mg/kg i.p.) and Dutasteride (40 or 80 mg/kg i.p.) were reported to reduce hyperlocomotion induced by D-amphetamine and attenuate stereotyped behaviors induced by the DA agonist apomorphine (Bortolato et al., 2008). In addition, it was reported that Finasteride administered at doses of 25 and 50 mg/kg i.p. to C57Bl/6 mice did not affect DA receptor agonists-induced behavior (Frau et al., 2013). 2.3. Preparation of brain tissue and assay of biogenic amines contents in brain tissue On Day 11, mice were decapitated; brains were quickly removed and frozen in isopentane (40  C) and trunk blood were collected in BD Vacutainer Venous Blood Collection Tubes. The left anterior striata were dissected and the DA and its metabolites 3,4dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) contents were measured by high performance liquid chromatography (HPLC) with electrochemical detection as previously described (D'Astous et al., 2003). 2.4. DAT and VMAT2 autoradiography The striatum (bregma 0.74e0.26 mm, according to the Atlas of Franklin and Paxinos Franklin and Paxinos, 2008) and the SNpc (bregma 2.92 to 3.64 mm) of the right hemisphere were cut on a cryostat in 12 mm slices and 6 consecutive brain sections were placed on a glass slide. Slices were kept at 80  C until assayed. DAT total binding used 20 pmol of the ligand 3b-(4-[125I] iodophenyl) tropane-2bcarboxylic acid isopropyl ester ([125I]-RTI-121) (2200 Ci/mmol; PerkinElmer, Boston, MA, USA). Non-specific binding was evaluated with binding in the presence of 100 nM of mazindol (Sandoz Pharmaceuticals, Dorval, Quebec, Canada) (Callier et al., 2001). Brain slices were apposed to Kodak BioMax MR films (Eastman Kodak Company, Rochester, NY, USA), 31 h for the striatum and 72 h for the SNpc. VMAT2 autoradiography (Kilbourn and Frey, 1996) was performed using the ligand [3H]dihydrotetrabenazine ([3H]-TBZ-OH) (20 Ci/mmol) and 1 mM of cold dihydrotetrabenazine was used to evaluate the nonspecific binding (American Radiolabeled Chemicals, St. Louis, MO, USA). Slices were exposed to Kodak BioMax MR films, 4 weeks for the striatum and 6 weeks for the SNpc. Films were analyzed using the software package NIH Image 1.63. The data presented shows the specific binding of the ligand that was calculated by subtracting the value for nonspecific binding from the total binding. 2.5. In situ hybridization of TH In situ hybridization of TH in the SNpc was performed as previously described (D'Astous et al., 2003). An oligonucleotide complementary to bases 1435e1482 (GenBank accession no. M69200) of the mice TH sequence was used and labeled at the 30 end with [33P]-dATP (3000 Ci/mmol, PerkineElmer, Waltham, MA, USA). The slide-mounted tissue sections were then exposed to Kodak BIOMAX film for 3.5 days at room temperature. The films were developed and the autoradiograms analyzed by densitometry using the software package NIH Image 1.63. The TH mRNA levels were expressed as optical densities. For each animal the mean of the total binding from 4 to 6 sections was measured. 2.6. Steroids assay The plasma steroid concentrations of T, DHT and 17b-estradiol were measured by GC/MS as described previously (Labrie et al., 2006). Plasma of 2 or 3 mice from the same group and with similar DA concentrations was pooled in order to have enough plasma to quantify the steroid concentrations. 2.7. Statistical analysis Statistical comparisons of the data were performed with analyses of variance using Stat View 4.51 software (Abacus Concepts, Inc., Berkeley, CA, USA) for Macintosh Computer (Apple Inc., Cupertino, CA, USA), followed by a post hoc

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Fig. 1. Schematic experimental design for administration of MPTP, vehicle (saline), Finasteride (5 or 12.5 mg/kg) or Dutasteride (5 or 12.5 mg/kg) and euthanasia. Male mice were administered intraperitoneal (i.p.) Finasteride, Dutasteride or vehicle once daily and received four intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6, tetrahydropyridine (MPTP) (4  6.5 mg/kg) on day 5. On the 5th day of treatment, the administration of Finasteride or Dutasteride was given an hour before the first injection of MPTP. analysis with a Fisher protected least significant difference test. A simple regression model was used to determine the coefficient of correlation. A p < 0.05 was required for the results to be considered statistically significant.

3. Results 3.1. Biogenic amines assay There was a statistically significant effect of treatments for DA (F5, 54 ¼ 9.66; p < 0.0001), DOPAC (F5, 54 ¼ 7.42; p < 0.0001), HVA (F5, 54 ¼ 4.40; p ¼ 0.002), DOPAC/DA (F5, 54 ¼ 4.42; p ¼ 0.0019), and HVA/DA (F5, 54 ¼ 8.51; p < 0.0001) ratios. A dose of MPTP was chosen to induce about 50% reduction in striatal DA contents in order to mimic an early stage of PD. Under these conditions there was no change in striatal 5-HT and its metabolite 5-HIAA contents after the MPTP lesion (Fig. 2). MPTP induced a significant reduction in striatal DA as well as its metabolites DOPAC and HVA contents compared to controls (Fig. 2). In MPTP mice treated with Dutasteride at 5 mg/kg and 12.5 mg/kg, a protective effect against MPTP

was observed on striatal DA and its metabolites contents. HVA and DOPAC striatal contents were significantly higher with the Dutasteride treatment compared to saline-treated MPTP mice; HVA remained close to control levels and DOPAC was about 80% of controls. By contrast, treatment with Finasteride at 5 or 12.5 mg/kg did not protect against MPTP toxicity striatal DA or its metabolites contents against MPTP toxicity. Finasteride treatments did not affect striatal 5-HT contents while it was slightly decreased with Dutasteride at 5 mg/kg. Striatal 5-HIAA contents were elevated in MPTP mice treated with both doses of Finasteride and Dutasteride. Mice treated with MPTP alone showed an increase in striatal DOPAC/DA ratio compared to controls that remained elevated with Finasteride treatments at 5 and 12.5 mg/kg. By contrast, Dutasteride treated MPTP mice had striatal DOPAC/DA ratio at control values (Dutasteride 5 mg/kg) and lower than for MPTP mice (Dutasteride at 12.5 mg/kg). Mice treated with MPTP also showed an increase in striatal HVA/DA ratio compared to controls that remained elevated with Finasteride treatment at 5 mg/kg and led to

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Fig. 2. Effects of a chronic treatment with Finasteride and Dutasteride on striatal dopamine (DA) contents and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), DOPAC/DA and HVA/DA ratios, as well as serotonin (5-HT) contents and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) of MPTP male mice compared to intact mice. Values shown are the means (ng/mg of proteins) ± standard error of the mean of 10 mice per group (number of mice in each group is included in each column of the graphs). *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 versus control; xp < 0.05, xxp < 0.01 versus MPTP; ##p < 0.01, ###p < 0.005, and ####p < 0.0001 versus MPTP þ Finasteride 12.5 mg/kg.

higher values compared to MPTP mice for Finasteride at 12.5 mg/ kg. By contrast, Dutasteride treated MPTP mice had striatal HVA/DA ratio at control values for both doses investigated.

3.2. Autoradiography of DAT and VMAT2 There was a statistically significant effect of treatments for striatal DAT (F5, 54 ¼ 7.660; P < 0.0001) and VMAT2 (F5, 53 ¼ 5.845; P ¼ 0.0002). MPTP treatment led to a significant decrease in striatal [125I]-RTI-121 specific binding to DAT with no change in the SNpc (Fig. 3). MPTP mice treated with Dutasteride

at 12.5 mg/kg, but not with Finasteride, showed striatal DAT levels significantly higher than vehicle-treated MPTP mice (Fig. 3). A significant decrease in striatal [3H]-TBZ-OH specific binding to VMAT2 was also observed following MPTP lesion with no change in the SNpc (Fig. 3). Dutasteride at 12.5 mg/kg prevented the decrease of striatal VMAT2 specific binding whereas Finasteride had no effect. A significant correlation between striatal specific binding to DAT and striatal DA contents as well as between VMAT2 and DA contents was observed (Fig. 4). A significant positive correlation was also observed between striatal DAT specific binding and VMAT2 specific binding.

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Fig. 3. Effects of a chronic treatment with Finasteride and Dutasteride on [125I]-RTI-121 dopamine (DA) transporter (DAT) and [3H]-dihydrotetrabenazine (TBZ-OH) vesicular monoamine transporter 2 (VMAT2) specific binding in striatum and SNpc and examples of these binding autoradiography of MPTP male mice compared to intact mice. Values shown are the means (fmol/mg of tissue) ± standard error of the mean of 8e10 mice per group (number of mice in each group is included in each column of the graphs). *p < 0.05, **p < 0.01 and ****p < 0.0001 versus control; xp < 0.05, xxp < 0.01 versus MPTP; ###p < 0.005 versus MPTP þ Finasteride 12.5 mg/kg.

3.3. In situ hybridization of TH

3.4. Steroids plasma concentrations

In situ hybridization analysis showed that TH mRNA levels remained unchanged in the SN (Fig. 5). This reflects the early phase of neurodegeneration where striatal DA terminals are degenerated while DA cell bodies in the SN are preserved.

In mice lesioned with MPTP, plasma T and DHT levels were significantly lower than in the vehicle-treated mice (Fig. 6). Administration of Finasteride and Dutasteride opposed the reduction of T induced by MPTP. A greater effect was observed with

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Fig. 4. Correlations between striatal dopamine (DA) contents with striatal [125I]-RTI-121 specific binding to dopamine transporter (DAT) and [3H]-TBZ-OH specific binding to vesicular monoamine transporter (VMAT2) as well as correlation between DAT with VMAT2 specific binding in Finasteride or Dutasteride and saline-treated MPTP male mice as well as in intact mice.

Dutasteride treatment at both doses where MPTP mice treated with Dutasteride 12.5 mg/kg had their T levels at control values. DHT plasma concentrations remained low in the Finasteride and Dutasteride treated MPTP mice. Levels of 17b-estradiol were under detection limits in the plasma of control mice and remained undetectable with the MPTP lesion and the treatments (Data not shown). 4. Discussion The present study, investigating Finasteride and Dutasteride in the MPTP mouse model of PD, showed that Dutasteride 1) prevented the MPTP toxicity on striatal DA contents, 2) prevented the DAT and VMAT2 loss in the striatum while Finasteride had no effect and 3) Dutasteride and Finasteride prevented the decrease in T plasma levels induced by MPTP. Mice were exposed to MPTP doses that induced a reduction of about 50% of striatal DA without affecting the cell bodies located in the SNpc as illustrated by TH mRNA levels as well as DAT and VMAT2 specific binding in MPTP mice. We showed that Dutasteride prevented the MPTP toxicity on DA metabolism. Dutasteride increased significantly striatal DA and its metabolites concentrations and decreased the rate of DA metabolism in MPTP-treated mice. By contrast, Finasteride did not prevent the decrease of striatal DA in MPTP mice. The decrease in DA turnover in the striatum following Dutasteride treatment may reflect a protection of the DA re-uptake mechanism of striatal terminals. The striatal 5HT content of these mice remained generally unchanged except for a small decrease with Dutasteride treatment at 5 mg/kg and increased 5-HIAA levels associated with VMAT2 specific binding at control levels, the only vesicular transporter implicated in storage ^me et al., 2011). Dutasteride at of 5-HT in the CNS (Narboux-Ne 12.5 mg/kg prevented the MPTP-induced decrease in VMAT2

specific binding, with 5-HT at control levels and increased striatal 5-HIAA levels suggesting increased 5-HT metabolism. Finasteride had no effect on striatal 5-HT contents while it increased 5-HIAA levels suggesting increased turnover. Degeneration of DA neuronal terminals is associated with a reduction in DA uptake and storage with impaired DAT and VMAT2 function (Harrington et al., 1996). Here, we found that Dutasteride treatments in MPTP mice prevented the DAT and VMAT2 loss in the striatum while Finasteride had no effect. Striatal DAT and VMAT2 specific binding positively correlated with DA concentrations as well as with each other. These correlations suggest that Dutasteride protected DA neuron terminals from neurodegeneration rather than promoted DA synthesis. Striatal DAT specific binding was significantly lower after Finasteride administration (12.5 mg/kg) compared to saline treated MPTP mice, suggesting that Finasteride could affect DAT differently than Dutasteride. More studies are needed to decipher cellular mechanisms involved in this phenomenon and to seek if the decrease in DAT specific binding is a direct or indirect effect of Finasteride. Genital dysfunction and T deficiency are non-motor symptoms reported in PD patients that respond favorably to T replacement therapy (Okun et al., 2002). MPTP administration in male mice was reported to induce a decrease of T plasma levels (Ruffoli et al., 2008) and a decrease of Leydig cells' number as well as ultrastructural alterations in the spared Leydig cells. These cells are the major site for producing endogenous T in physiological conditions (Mayerhofer et al., 1989). In the present study MPTP led to a reduction of plasma T and DHT levels as we previously reported in MPTP mice (Bourque et al., 2014). Treatment of MPTP mice with 12.5 mg/kg Dutasteride completely opposed the decrease in T plasma levels induced by the lesion whereas Finasteride at this dose led to a less significant decrease compared to Dutasteride. Hence, our findings showed that the MPTP model mimics the T

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Fig. 5. Effects of a chronic treatment with Finasteride and Dutasteride on Tyrosine Hydroxylase mRNA levels in the SNpc and examples of these in situ hybridization of MPTP male mice compared to intact mice. Results are expressed in relative optical density ± standard error of the mean of 6e8 mice per group (number of mice in each group is included in each column of the graphs).

Fig. 6. Effects of a chronic treatment with Finasteride and Dutasteride on plasma levels of testosterone and dihydrotestosterone of MPTP male mice compared to intact mice. Values shown are the means ± standard error of the mean. *p < 0.05, **p < 0.01 and ****p < 0.0001 versus control.

hormone deficiency observed in PD patients, which could be very useful to study the effect of potential neuroprotective drugs as Dutasteride on non-motor functions. The mechanism involved in the neuroprotective effect of Dutasteride is presently unknown. 5a-reductase inhibition by blocking the metabolism of T and P to its metabolites, could lead to accumulation of precursors and some of them have shown neuroprotective activity in MPTP mice such as estrogens, P and dehydroepiandrosterone (DHEA) (Bourque et al., 2009) (Callier et al., 2001). Given that the P metabolite AP also exhibits protective

activity in MPTP mice (Adeosun et al., 2012), neuroprotective effects could be due to activation of P receptor by P and DHP, and also to activation of the g-aminobutyric acid type A (GABA-A) receptor by AP (Belelli and Lambert, 2005). The neuroprotective effects observed here are not likely to be mediated by GABA-A activation by AP since this metabolite is likely decreased due to reduction of P metabolism by 5a-reductase inhibition. Moreover, increased T levels by inhibition of its metabolism with a 5a-reductase inhibitor can be converted to estradiol via aromatases, which are expressed in the striatum (Küppers et al., 2000). The lack of effect of T and the

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non-aromatizable androgen, DHT, to protect DA neurons against MPTP toxicity suggests that the neuroprotective effect of Dutasteride could be through the aromatization of T to 17b-estradiol. However, plasma levels of 17b-estradiol in males are low and were undetectable in the plasma of male mice of the present experiment. In addition, both Finasteride and Dutasteride inhibit transformation of T to DHT that could be redirected to the formation of 17b-estradiol but only Dutasteride was shown here to be neuroprotective. PD pathology is considered to involve multiple factors with interactions between genetic predisposition and environmental exposure (Gao and Hong, 2011). Thus, in order to treat motor and non-motor symptoms of PD, multi-target drugs may be more appropriate (Youdim et al., 2014). Therefore, Dutasteride appears to be a promising drug as it acts, by its inhibitory activity on 5areductase, on the modulation of neurosteroids synthesis and/or metabolism and implies also many other molecular mechanisms in the neuroprotective actions. 5a-reductase inhibitors could be neuroprotective against DA MPTP toxicity by affecting neurosteroid levels acting on receptors involved in neuroprotection such as sigma-1 receptors (Francardo et al., 2014; Su et al., 1988) since various steroids, such as P, T, pregnenolone sulfate and DHEA, have affinity for sigma-1 receptors. However, these steroids function as agonists or antagonists and their combined effect on sigma-1 receptors is yet to be measured (Hayashi and Su, 2004). Dutasteride could also protect striatal DA by directly or indirectly affecting mitochondria function since many studies report impaired mitochondrial dysfunction in PD (Winklhofer and Haass, 2010). Accordingly, in vitro Finasteride and Dutasteride were reported to protect neural cells from death induced by transient intracellular calcium concentration surges (Soski
c et al., 2008). In addition in vivo, Dutasteride is shown to inhibit in male mice the formation of b-amyloid plaque load in a model of cerebral amyloidosis, which seems to link mitochondrial apoptosis and autophagy (Soski
c et al., 2008). The prevalent 5a-reductase isoenzyme in the adult brain has been reported to be of type I (Stoffel-Wagner, 2003) which is responsible for the synthesis of neuroactive steroids in the brain (Negri-Cesi et al., 1996) including the medium spiny neurons in the striatum (Agís-Balboa et al., 2006). Finasteride is a 5a-reductase inhibitor of the human types I and II with more than 100 times selectivity for human type II (Bull et al., 1996). By contrast in rats, Finasteride is a potent inhibitor of both types I and II 5a-reductases (Azzolina et al., 1997; Thigpen and Russell, 1992) thus limiting the identification of the relative implication of each isoform in the metabolism of neurosteroids and the assessment of the molecular basis of the neuroprotective effect. Furthermore, 5a-reductase expression is reported to be increased in the presence of DHT (George et al., 1991). A previous study reported that both 5areductase isoenzymes are regulated positively by androgens with significantly decreased mRNA levels of both 5a-reductases in castrated rats. Previous experiments showed that type I 5a-reductase was regulated positively by androgens in the liver of male rat and its expression increased in both intact and castrated animals after T and DHT treatment (Torres et al., 2003). Thus, DHT seems to play a key role in the increase of type I 5a-reductase mRNA levels; partial inhibition of activity of this enzyme by Finasteride allows yet the synthesis of DHT by type II 5a-reductase that in turn regulates positively the enzyme activity. This mechanism of feed-forward stimulation may unbalance the action of Finasteride insofar as the 5a-reductase will be stimulated. The lack of DA neuroprotection observed here with Finasteride could also be due to its short serum half-life compared to Dutasteride. In rats, serum half-life of Dutasteride is reported to be 31 h and 2 h for Finasteride (Xu et al., 2006). Hence, compared to

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Finasteride, Dutasteride injected once daily, given its affinity and long half-life, would exhibit a more sustained protection to counteract MPTP toxicity. This could be offset with higher doses of Finasteride to extend its activity as previously reported (Bortolato et al., 2008) but would be associated with reduced motor activity (Frau et al., 2013). In conclusion, the main finding of this study is that the 5areductase inhibitor Dutasteride but not Finasteride protected DA neurons against MPTP toxicity. This is the first report of a neuroprotective activity of a 5a-reductase inhibitor in an in vivo animal model of PD. More studies are required to investigate the molecular mechanisms implicated in this neuroprotection and to study the neurorescue and neurorestorative properties of Dutasteride. These results in MPTP mice have shown that 5a-reductase enzymes could be interesting novel targets for new therapies for the treatment of PD. Conflict of interest All authors declare that they have no conflicts of interest. Role of the funding source The funding sources had no role on the study design, the collection, analysis, and interpretation of the data, nor in writing the manuscript or the decision on where to submit the manuscript for publication. Acknowledgments This research was supported by a grant from the Canadian Institutes of Health Research (CIHR-grant number MOP-82692 e Canada) to TDP, a PhD scholarship from the Faculty of Pharmacy of Laval University (Canada) and a Graduate Student Award from the Parkinson Society Canada (Canada) to NL. References Adeosun, S.O., Hou, X., Jiao, Y., Zheng, B., Henry, S., Hill, R., He, Z., Pani, A., Kyle, P., Ou, X., Mosley, T., Farley, J.M., Stockmeier, C., Paul, I., Bigler, S., Brinton, R.D., Smeyne, R., Wang, J.M., 2012. Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson's disease. PLoS ONE 7, e50040. http://dx.doi.org/10.1371/ journal.pone.0050040. Agís-Balboa, R.C., Pinna, G., Zhubi, A., Maloku, E., Veldic, M., Costa, E., Guidotti, A., 2006. Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 103, 14602e14607. http://dx.doi.org/10.1073/pnas.0606544103. Azzolina, B., Ellsworth, K., Andersson, S., Geissler, W., Bull, H.G., Harris, G.S., 1997. Inhibition of rat a-reductases by finasteride: evidence for isozyme differences in the mechanism of inhibition. J. Steroid Biochem. Mol. Biol. 61, 55e64. http:// dx.doi.org/10.1016/S0960-0760(97)00002-2. Becker, J.B., 1999. Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol. Biochem. Behav. 64, 803e812. http:// dx.doi.org/10.1016/S0091-3057(99)00168-9. Belelli, D., Lambert, J.J., 2005. Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat. Rev. Neurosci. 6, 565e575. http://dx.doi.org/10.1038/nrn1703. Bortolato, M., Frau, R., Orrù, M., Bourov, Y., Marrosu, F., Mereu, G., Devoto, P., Gessa, G.L., 2008. Antipsychotic-like properties of 5-alpha-reductase inhibitors. Neuropsychopharmacology 33, 3146e3156. http://dx.doi.org/10.1038/npp. 2008.39. Bourque, M., Dluzen, D.E., Di Paolo, T., 2009. Neuroprotective actions of sex steroids in Parkinson's disease. Front. Neuroendocrinol. 30, 142e157. http://dx.doi.org/ 10.1016/j.yfrne.2009.04.014. Bourque, M., Morissette, M., Di Paolo, T., 2014. Raloxifene activates G proteincoupled estrogen receptor 1/Akt signaling to protect dopamine neurons in 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice. Neurobiol. Aging 35, 2347e2356. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.017. Bull, H.G., Garcia-Calvo, M., Andersson, S., Baginsky, W.F., Chan, H.K., Ellsworth, D.E., Miller, R.R., Stearns, R.A., Bakshi, R.K., Rasmusson, G.H., Tolman, R.L., Myers, R.W., Kozarich, J.W., Harris, G.S., 1996. Mechanism-based inhibition of human steroid 5a-reductase by Finasteride: enzyme-catalyzed formation of

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