Neurotoxicity Research https://doi.org/10.1007/s12640-017-9853-3

ORIGINAL ARTICLE

Peripheral Administration of Tetanus Toxin Hc Fragment Prevents MPP+ Toxicity In Vivo Natalia Moreno-Galarza 1 & Liliana Mendieta 2 & Victoria Palafox-Sánchez 2 & Mireia Herrando-Grabulosa 1 & Carles Gil 1 & Daniel I. Limón 2 & José Aguilera 1,3 Received: 26 June 2017 / Revised: 8 December 2017 / Accepted: 11 December 2017 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Several studies have shown that intrastriatal application of 1-methyl-4-phenylpyridinium (MPP+) produces similar biochemical changes in rat to those seen in Parkinson’s disease (PD), such as dopaminergic terminal degeneration and consequent appearance of motor deficits, making the MPP+ lesion a widely used model of parkinsonism in rodents. Previous results from our group have shown a neuroprotective effect of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) under different types of stress. In the present study, pretreatment with the intraperitoneal injection of Hc-TeTx in rats prevents the decrease of tyrosine hydroxylase immunoreactivity in the striatum due to injury with MPP+, when applied stereotaxically in the striatum. Similarly, striatal catecholamine contents are restored, as well as the levels of two other dopaminergic markers, the dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT-2). Additionally, uptake studies of [3H]-dopamine and [3H]-MPP+ reveal that DAT action is not affected by Hc-TeTx, discarding a protective effect due to a reduced entry of MPP+ into nerve terminals. Behavioral assessments show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP+-treated rats. Our results lead us to consider Hc-TeTx as a potential therapeutic tool in pathologies caused by impairment of dopaminergic innervation in the striatum, as is the case of PD. Keywords Carboxyl-terminal domain of tetanus toxin . Parkinson’s disease . Tyrosine hydroxylase . Dopamine . Neuroprotection . 1-Methyl-4-phenylpyridinium

Introduction Parkinson’s disease (PD) is the second most common agerelated neurodegenerative disorder, characterized by a progressive degeneration of the dopaminergic neurons located in the substantia nigra pars compacta (SNpc), with a concomitant loss of dopamine (DA) content in the

* José Aguilera [email protected] 1

Institut de Neurociències and Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain

2

Laboratorio de Neurofarmacología, FCQ-Benemérita Universidad Autónoma de Puebla, Puebla, Mexico

3

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Cerdanyola del Vallès, Barcelona, Spain

striatum (nucleus caudate and putamen). DA deficiency is responsible for the major motor symptoms of PD, which include resting tremor, bradykinesia, postural instability, and rigidity (Ferrer 2011). Although the molecular events that originate the disease remain unclear, different palliative treatments are used to alleviate most of these symptoms, with the administration of levodopa being the most effective treatment in the first years of disease. At that point, adverse reactions such as dyskinesia and on-off and wearing-off phenomena appear (Ahlskog and Muenter 2001). Unfortunately, all current existing therapies, medical and surgical, are symptomatic and none of them is able to prevent the dopaminergic neuron degeneration (Choonara et al. 2009; Schapira et al. 2014). In PD, striatal dopaminergic nerve terminals appear to be the primary target of the degenerative process and their destruction precedes SNpc neuronal death, which occurs in a Bdying back^ manner (Aron et al. 2010). In vivo approaches with the neurotoxin 1-methyl-4-phenyl-

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1,2,3,6-tetrahydropyridine (MPTP) reproduce these observations, reinforcing the retrograde neuronal death theory (Herkenham et al. 1991). This compound, MPTP, has been shown to act as a proneurotoxin targeting DA neurons and causing parkinsonism in humans, nonhuman primates, rodents, and in other nonmammalian species (Banerjee et al. 2006). In the brain, MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP+), the active toxic molecule, which enters the dopaminergic neurons through the dopamine transporter (DAT) and acts on the mitochondria, impairing the multienzyme complex I of the mitochondrial electron transport chain (Nicklas et al. 1985; Annepu and Ravindranath 2000). This blockade is responsible for an energetic crisis, inflammation, and oxidative stress (Ferrer 2011; Przedborski and Vila 2003) that finally cause death cell. In the present study, we optimized a modification of a published PD model (Ghorayeb et al. 2002), adjusting the injected MPP+ dose into the nucleus striatum to obtain an animal model in which DA terminals in the striatum degenerate and, in consequence, low DA content is found, without eliciting neuronal death in the SNpc (Fallon et al. 1997). Thus, the aim of this protocol is to mimic the first events, which take place at initial nonsymptomatic stages of PD, and not to induce a PD model in advanced stage, i.e., with death of the dopaminergic bodies located in the SNpc. In this regard, MPP+ intrastriatal has been used to test potential anti-Parkinsonian drugs in rats (Geed et al. 2014; PérezBarrón et al. 2015). On the other hand, the nontoxic carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) has been used as a valuable tool in molecule delivery into the central nervous system (CNS), because of its efficient retroaxonal transport (Cordero-Erausquin et al. 2009; Li et al. 2009; Larsen et al. 2006). Our group has repeatedly published the neurophysiological similarity between TeTx (or its Hc-TeTx fragment) and some growth factors (e.g., NGF and BDNF), triggering the same signal transduction pathways and responding similarly to different stresses and in different cellular systems (Gil et al. 2001, 2003). Moreover, Hc-TeTx elicits neuroprotection of cerebellar granular cells (CGCs) against apoptotic death caused by potassium deprivation (Chaïb-Oukadour et al. 2004) or by the mitochondrial poison MPP + (ChaïbOukadour et al. 2009). In vivo, Hc-TeTx ameliorates motor behavior and improves the dopaminergic striatal system of rats with MPP+ lesions, when co-administrated with the neurotoxin (Mendieta et al. 2009). The present work explored the ability of peripherally injected Hc-TeTx to prevent, in vivo, the neurotoxic action of MPP+, to evaluate the potential use of Hc-TeTx as a molecule with benefits in the treatment against the progression of degenerative diseases in which dopamine terminals are selectively affected, like in PD.

Material and Methods Animals Male Sprague-Dawley rats aged 8 weeks and weighing 250– 300 g were used. Rats were randomly divided into four groups: control, Hc-TeTx, MPP+, and Hc/MPP+. All determinations were obtained under licenses issued by the Ethical Committee of the Universitat Autònoma de Barcelona. Also, the Mexican Council for Animal Use and Care from Benemerita Universidad Autonoma Puebla has been followed.

Expression and Purification of Hc-TeTx The recombinant carboxyl-terminal domain of the heavy chain of tetanus toxin, Hc-TeTx fragment or Hc fragment, was synthesized in accordance to Chaïb-Oukadour et al. (2004). At the onset of the experiments (day 0) and at day 3, Hc-TeTx and Hc/ MPP+ rats received an intraperitoneal (i.p.) injection of HcTeTx (0.5 mg/kg) dissolved in isotonic solution, whereas the control and MPP+ rats received an i.p. injection of vehicle.

Intrastriatal Injections At day 7, all rats underwent stereotaxic surgery (David Kopf instruments, Tujunga, CA, USA). Animals received, 15 min prior to surgery, a dose of desipramine (25 mg/kg, i.p.), in order to avoid nonspecific uptake of MPP+ through the noradrenergic transporters (NET) (Barc et al. 2002). Animals were anesthetized using isoflurane, and the control and HcTeTx animals were infused with vehicle, while rats from the MPP+ and Hc/MPP+ groups were infused with 10 μg of MPP+ (Sigma, St. Louis, MO, USA) using the following stereotaxic coordinates: AP:+0.7 mm from Bregma, L: -2.8 mm from midline, DV:-6.0 below dura in according to Paxinos and Watson’s Stereotaxic Atlas (1998). Proper post-operative attention was provided until the animal made a full recovery. Animals were sacrificed at days 0, 3, 5, 7, or 9 after the intrastriatal infusion in the case of dopamine system evaluation with high-performance liquid chromatography with electrochemical detection (HPLC-EC). In the case of animals destined to Western blot and immunohistochemistry experiments, they were sacrificed 5 days after the intrastriatal infusion.

Determination of Dopamine and Its Metabolites by High-Performance Liquid Chromatography with Electrochemical Detection Sprague-Dawley rats were sacrificed and their nuclei striatal were used to determine the endogenous levels of DA, 3,4dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT). Tissues were dissected, weighted, and frozen in isopentane. Thereafter, they

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were homogenized by sonication in 10 volumes (w/v) of 0.25 M perchloric acid containing 250 μM EDTA-Na2, 100 μM sodium metabisulfite, and 1000 ng/mL 3,4dihydroxybenzylamine (DHBA), as an internal standard for catecholamine determination. The homogenates were stored at − 80 °C. At the time of analysis, samples were analyzed by HPLC-EC, as described elsewhere (Mendieta et al. 2009).

performed with the ImageJ free software from NIH. The nucleus striatum was delineated as follows: The outer border of the striatum was defined by the lateral ventricle medially, the corpus callosum dorsally and laterally, and by a line drawn between the anterior commissure and the corpus callosum ventrally. The OD was measured as the integrated density of the mean gray values per measured area. TH-IR OD was measured bilaterally and arbitrary units were assigned.

Immunohistochemistry Rats were anesthetized with isofluorane and perfused transcardially with 4% paraformaldehyde. Brains were removed, postfixed in the same fixative solution for 48 h, and transferred into 30% sucrose in PBS for 48 h. Coronal sections of 30 μm for nucleus striatum and 20 μm for substantia nigra were cut with a cryostat cryotome (Thermo Electron Corporation) and conserved at − 20 °C. Every six serial sections were washed three times in Trisbuffered saline (TBS pH 7.6) for 20 min, once in TBS supplemented with 0.2% Triton X-100 (TBST) and then blocked in blocking solution (TBST supplemented with 5% bovine serum albumin, BSA) for 1 h at room temperature. Then, sections were incubated overnight at 4 °C with monoclonal antibody antityrosine hydroxylase Clone TH-16 (Sigma) at 1:1250 dilution in TBST and 2.5% BSA. After that, the sections were incubated for 15 min in 20% methanol and 2% H2O2 in TBST, in order to quench endogenous peroxidase activity. Thereafter, the sections were incubated in the secondary antibody rat anti-mouse biotinylated antibody (1:200; Amersham Biosciences, UK) in TBST and 5% BSA for 1 h at room temperature, after which the sections were incubated in avidin-HRP complex (1:400; Dako Cytomation, Denmark), TBST, and 2.5% BSA for 1 h at room temperature. The sections were gently washed with TBS between all the described steps. TH immunoreactivity was visualized by 3,3′-diaminobenzidine tetrachloride (DAB, Sigma) reaction in the striatum and substantia nigra after an incubation of 6–9 min. To verify the specificity of the immunostaining, control sections were processed in an identical manner, but omitting the primary antibody. Sections were gently washed in TB, mounted on slides, dried, dehydrated in graded ethanol, cleared in xylol, and coverslipped with DPX medium.

Quantification of Tyrosine Hydroxylase Immunoreactivity in the Nucleus Striatum Striatal dopaminergic innervation was quantified as described in Zhu et al. (2004) with slight modifications. The optical density (OD) of tyrosine hydroxylase immunoreactivity (TH-IR) was measured in 1 of every 6 sections (12–15 sections per animal). These sections were captured by a digital camera (Nikon DXM 1200F) connected to a microscope (Nikon Eclipse 90i) coupled to a computer (Software Nikon ACT-1), with a ×2 objective. The analysis of the images was

Quantification of Tyrosine Hydroxylase-Positive Cells in Substantia Nigra Pars Compacta The number of TH-positive immunoreactive neurons in the substantia nigra pars compacta (SNpc) was determined as described by Liu et al. (2007) with slight modifications. One every 6th sections (10 sections) were counted under a light microscope, covering the entire extent of the SNpc with a ×40 objective. The neurons of both hemispheres, ipsi- and contralateral to the lesion, were counted.

Western Blot Five days after stereotaxic infusion, striatal tissue was dissected and placed in nine volumes of homogenization buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 50 mM NaF, 0.5% sodium deoxycolate, 0.1% SDS, 0.5% Triton X-100, 2.5 mM EDTA, 10 μg/mL leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate) using a Potter homogenizer. Samples containing equal amounts of protein (20 μg) were analyzed by Western blot, as described in Gil et al. (2003). Antibodies were monoclonal anti-tyrosine hydroxylase clone TH-16 (Sigma 1:2000), rabbit polyclonal anti-DAT (Santa Cruz 1:50), rabbit polyclonal antiVMAT-2 (Chemicon 1:500), and monoclonal anti-β-actin (Sigma 1:10,000).

Dopamine and MPP+ Uptake of Rat Striatal Synaptosomes Striatal tissue was extracted and weighted and synaptosomes were obtained as previously described (Gil et al. 2005). Before the uptake procedure, synaptosomes were tempered for 30 min at 37 °C in a shaking water bath. In the standard assay to measure synaptosomal dopamine uptake, 50 μL aliquots of synaptosomal fraction were pretreated in a polypropylene tube with Hc-TeTx at final concentrations of 1, 10, or 100 nM, for 15 min. The uptake was started by the addition of 50 μL of dopamine or vehicle. Uptake was assayed using tritiated dopamine (3[H]-DA, 10 nM) and nonradiolabeled dopamine to obtain final concentrations in the 10- to 640-nM range in the case of saturation experiments. Nonspecific dopamine uptake was performed at 0–4 °C. The general procedure followed in the MPP+ uptake was the same as in the case of dopamine,

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using a MPP+ concentration range from 10 to 1500 nM. Uptake was assayed using tritiated MPP + ( 3[H]-MPP +, 10 nM) and nonradiolabeled MPP+ to obtain the desired final concentrations. The pretreatment with Hc-TeTx was performed at 100 nM final concentration. The dopamine or the MPP+ uptake experiments were terminated after 2 min by filtration through Whatman GF/C filters presoaked in polyethylenimine 0.3% in a vacuum filtration manifold (Brandel Inc.) and washed three times with 5 mL of ice-cold Krebs-Ringer bicarbonate buffer. Filters were then dried, placed in vials containing 5 mL of Biodegradable Counting Scintillant (Amersham International) and counted in a Wallac-1409 liquid scintillation counter. Radioactivity accumulated by synaptosomes at 0–4 °C was routinely subtracted, and temperature-dependent uptake was defined as the difference between the uptake carried out at 37 and at 4 °C. Each reaction was performed in triplicate or quadruplicate and the uptakes were assayed 3–5 times. Data were analyzed by nonlinear regression using the curve fitting program GraphPad Prism 5 Software Version 5.0.

Assessment of Movement Skills The animals were tested for rotational behavior for 80 min after methamphetamine injection (5.0 mg/kg s.c.; Sigma, St. Louis, MO, USA) on day 5 after stereotaxic injection in all groups, as described (Ungerstedt and Arbuthnott 1970). Forelimb akinesia was assessed using the cylinder test, as described (Schallert et al. 2000). In the adjusting step test, the animals were tested during the third week after lesion, as described elsewhere (Olsson et al. 1995). Briefly, rats were held at the rear part of the torso by one hand with their hindlimbs lifted, and one forepaw was held steady with another hand of the experimenter so as to bear weight on the other forepaw. The animals performed the stepping test on a surface for 1 m, at a speed of about 14.0 cm/s. Each stepping test consisted of two trials for each forepaw, alternating between forepaws. Three days before the dopaminergic lesion, all of the animals were assessed for the stepping test to obtain the basal values. The same procedure was carried out in the three consecutive weeks after the lesion (7, 12, 17, and 22 days postlesion). Each session was recorded by a video camera, and the single values calculated for each rat were used to obtain the group average.

differences, a Bonferroni post hoc test was applied. Statistical significance was designated at p > 0.05. Statistical analyses were performed using GraphPad Prism 5 Software Version 5.0 for Windows (GraphPad Software).

Results The Intrastriatal Infusion of a Low Dose of MPP+ Causes a Transient Loss of Dopaminergic Metabolites in Rat Striatum To determine the effects of a low dose of MPP+ on the dopamine and related metabolite contents in the striatum, male Sprague-Dawley rats were infused unilaterally in the right striatum with 10 μg of the neurotoxin MPP+ and were sacrificed at days 0, 3, 5, 7, or 9 after infusion, as stated in the BMaterials and Methods^ section. The HPLC analysis of the striatal homogenates showed that the MPP+ infusion caused a significant reduction of DA concentration in the injected striatum on day 3 (p < 0.01) and reached the maximal depletion on day 5 (p < 0.001), where the DA concentration represented about 50% of the control. On days 7 and 9, striatal DA levels were recovered, reaching similar levels to those from the control (p > 0.05), (Fig. 1a). The results from the DOPAC, HVA, and 3-MT metabolites (Fig. 1b, c and d, respectively) also showed transient decreases after MPP+ infusion.

Hc-TeTx Pretreatment Prevents the Loss of Dopaminergic Metabolites Caused by MPP+ Male Sprague-Dawley rats were intraperitoneally injected with Hc-TeTx (0.5 mg/kg) or with vehicle twice, on days 0 and 3. On day 7, stereotaxic infusion of MPP+ (10 μg) or of vehicle was performed in the right striatum, and rats were sacrificed on day 5 after the stereotaxic injection, a time that corresponds to the period of maximal effect, as described in Fig. 1. Dopaminergic levels in striatal homogenates were analyzed by HPLC (Fig. 2). Pretreatment with Hc-TeTx totally prevented the decrease in the dopamine, DOPAC, homovanillic acid, and 3-methoxytyramine contents exerted by MPP+, whereas Hc-TeTx injected alone did not produce any significant changes in DA or metabolite levels. In the contralateral hemisphere of the brain, no significant changes were found.

Statistical Analysis Statistical analyses were performed using Student’s t test for two mean comparisons or one-way analysis of variance (ANOVA) for more than two means comparison. For behavioral analyses, one-way ANOVA for repeated measures test were performed. When ANOVA showed significant

Hc-TeTx Does Not Alter 3[H]-DA or 3[H]-MPP+ Uptakes in Rat Striatal Synaptosomal Preparations Since MPP+ neurotoxin access into dopaminergic neurons depends on the plasma membrane dopamine transporter (DAT) activity (Dietz et al. 2008) and our group has

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Fig. 1 Time-course of the effect exerted by MPP+ on the striatal content of dopamine and dopaminergic metabolites. Following the MPP + intrastriatal administration (10 μg), the levels of DA (a) and metabolites DOPAC (b), HVA (c), and 3-MT (d) were analyzed after 0, 3, 5, 7 and 9 days, by means of HPLC coupled to an electrochemical detection system. The graphics show a highly significant decrease in DA levels on day 5 and a visible recovery on day 7. The striatal concentrations of DOPAC, HVA, and 3-MT follow a similar temporal pattern. Noradrenaline (NA)

levels in the striatum were also measured, but no differences were found (data not shown). In parallel, the same experiment was performed with hypothalamic tissue, and DA, DOPAC, and NA levels were analyzed. In this case, no changes were detected (data not shown). Data are mean ± SEM of three independent experiments. n = 3–4 per group and experiment (***p < 0.001, **p < 0.01). (DA, dopamine; DOPAC, 3,4dihydroxyphenylacetic acid; HVA, homovanillic acid; and 3-MT, 3methoxytyramine)

Fig. 2 Determination of dopamine and dopaminergic metabolites DA (a), DOPAC (b), HVA (c), and 3-MT (d), by HPLC 5 days after MPP+ infusion. The treatment with 10 μg of MPP+ applied intrastriatally produces a reduction in the levels of striatal dopamine and its metabolites in the ipsilateral side of the lesion, after 5 days. This decrease in dopamine and its metabolites is totally prevented by Hc-

TeTx (0.5 mg/kg), when injected intraperitoneally previous to MPP+ application. The data are expressed as the mean ± SEM of four independent experiments (n = 3–4 animals per group and experiment). (*p < 0.05, **p < 0.01, vs control). No significant variations were observed in the contralateral hemisphere

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previously demonstrated that the Hc-TeTx fragment alters the 5-HT uptake through its SERT transporter (Inserte et al. 1999), which is structurally and functionally similar to the DAT transporter, we decided to test whether the Hc-TeTx fragment exerted some effect on the kinetic parameters (Km and Vmax) of the DAT transporter. In Fig. 3a, the effect of Hc-TeTx pretreatment on the DA uptake of synaptosomally enriched fractions (P2) from rat striatum is shown in saturation curves, demonstrating that pretreatment with 1, 10, or 100 nM HcTeTx failed to modify the 3[H]-DA uptake parameters through the DAT transporter (table in Fig. 3b). Statistical analysis revealed no significant differences between the apparent values of the kinetic parameters, Km and Vmax, of the treated groups versus the control group (p > 0.05). To determine if Hc-TeTx was able to modify the 3[H]MPP+ uptake through the DAT transporter, striatal synaptosomes were pretreated with Hc-TeTx (100 nM final concentration). The statistical analysis of the saturation curves showed no significant differences in the kinetic parameters between Hc-TeTx-treated synaptosomes and vehicle-treated synaptosomes (p > 0.05) (Fig. 3b and table). These data demonstrate that Hc-TeTx is not able to change the Km or Vmax kinetic parameters of the DAT transporter and, consequently, does not change DA or MPP+ uptake through the DAT transporter in striatum preparations.

Hc-TeTx Prevents the Loss of Dopaminergic Innervation in the Striatum Caused by MPP+ In order to determine the effect of the different treatments on dopaminergic innervations in the striatum, the quantification of TH immunoreactivity was performed in coronal sections from the brains 5 days after the unilateral vehicle or MPP+ intrastriatal infusion, as stated in the “Materials and Methods.” Intrastriatal infusion of vehicle (1 μL) did not cause any difference in TH immunostaining, neither in the left nor in the right striatum (Fig. 4a). On the contrary, MPP+ unilateral administration (10 μg in 1 μL vehicle) led to loss of TH immunostaining of about 40% in the ipsilateral striatum (*p < 0.05). Intraperitoneal pretreatment with Hc-TeTx (0.5 mg/kg) prior to the intrastriatal MPP+ injection prevented the decrease of the TH signal, the obtained OD values not being significantly different from those found for the control group. The rats pretreated with Hc-TeTx (i.p.), but injected intracranially with vehicle (1 μL) instead of MPP+, did not suffer any significant change in the TH immunoreactivity of the ipsilateral striatum. In all groups, the contralateral TH immunostaining was comparable to that of the control group. In Fig. 4b, the graphic shows the observed OD of TH for both the ipsilateral and contralateral brain hemispheres of all experimental groups, with OD being expressed in percentage of the control. These results show that the i.p. treatment with Hc-TeTx was able to prevent the loss of dopaminergic innervations in the striatum

Fig. 3 Effect of Hc-TeTx on dopamine uptake and on MPP+ uptake in rat striatal synaptosomes. a Synaptosome aliquots were pretreated for 15 min at 37 °C with different Hc-TeTx concentrations (0, 1, 10, or 100 nM) and subsequently incubated for 2 additional min at 37 °C with dopamine over a concentration range from 10 to 640 nM, as described in the BMaterials and Methods.^ b After 15 min pretreatment with Hc-TeTx (100 nM), striatal synaptosomes were incubated for 2 additional min at 37 °C with MPP+, over a concentration range from 10 to 1500 nM. Results are expressed as the specific DA (a) or MPP+ uptake (b), mean ± SEM of five (n = 5) and three (n = 3) independent experiments, respectively. Saturation curves of treated synaptosomes did not differ significantly from those of the control group (p > 0.05). The tables show the apparent V max and K m values obtained after each treatment. In consequence, the Hc-TeTx fragment did not modify the kinetic parameters of dopamine or of MPP+ uptake. n.s., not significant

due to MPP+ insult. In order to confirm the aforementioned results observed in the immunohistochemistry assays,

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TH, and VMAT-2 and β-actin are shown, for the ipsilateral and contralateral sides of the brain. A graph under each blot represents the OD values in percentage obtained for each experimental group.

MPP+ Applied at Low Dose in the Striatum Is not Able to Reduce the Number of TH-Positive Neurons in the Substantia Nigra Pars Compacta

Fig. 4 Quantification of dopaminergic innervation in the striatum 5 days after unilateral vehicle or MPP+ intrastriatal infusion. Coronal brain sections (30 μm) were immunostained for TH. a Representative images of control, Hc-TeTx, MPP+, and Hc/MPP+-treated rats. The ipsilateral side was slightly cut off at the bottom to facilitate its identification (dotted line in the control image). An example of an area whose OD was measured as stated in the BMaterials and Methods^ is shown in the control image. b The intrastriatal injection of MPP+ causes a significant and remarkable decrease of TH in the ipsilateral side of the brain. Pretreatment with Hc-TeTx partially restores the TH levels in the striatum. Results are expressed as the mean values ± SEM obtained in five separate experiments. n = 3–4 per group and experiment (*p < 0.05)

analyses by Western blot of TH, as well as of DAT and of vesicular monoamine transporter 2 (VMAT-2), were also performed in the same conditions as those in Fig. 4. Thus, on the fifth day after intrastriatal MPP+ or vehicle injection, both striatal nuclei were extracted and homogenized separately and Western blots were performed (Fig. 5). In all cases, samples from rats treated with MPP+ alone showed significant decreases in DAT, TH, and VMAT-2 levels of approximately 50%, with respect to the control group. Pretreatment with HcTeTx (i.p.) prevented these decreases in the ipsilateral side of the lesion. In rats only treated with Hc-TeTx, no significant differences were found (p > 0.05) in the content of dopaminergic markers, as compared to the control group. Additionally, no significant changes were observed in the contralateral hemispheres. In Fig. 5, representative blots of DAT,

Since the dopaminergic terminals reaching the striatum are described to have their neuronal bodies in the SNpc (Prensa and Parent 2001), in the so-called nigrostriatal pathway, TH immunoreactivity was also determined in the SNpc (Fig. 6) in order to assess whether the loss of DA terminals in the striatum was due to DA neuronal death in SNpc. Figure 6a shows coronal sections containing the SNpc and VTA of each experimental group, and Fig. 6b shows a representative image of SNpc at ×40 magnification. As is shown in the graph (Fig. 6c), the counting of positive TH neurons in the SNpc, after THspecific immunohistochemistry, revealed no significant differences between the number of TH-positive cells found in the control group (1326 ± 59), with respect to the MPP+-injected rats (1158 ± 68) (ipsilateral sides). The same result was obtained when the Hc-TeTx group (1126 ± 41) and Hc/MPP+ group (1234 ± 132) were compared with the respective contralateral sides or with the control group. The absence of degenerating neurons in SNpc after MPP+ treatment was also confirmed by Fluoro-Jade B staining (data not shown). Thus, the degeneration of the dopaminergic terminals contained in the striatum triggered by the infusion of MPP+ did not cause the retrograde death of the neuronal bodies contained in the SNpc, the MPP+ effect only being local at the site of application, i.e., in the striatum, in the conditions of the presented assays.

Hc-TeTx Pretreatment Prevents the Movement Impairment Caused by MPP+ On day 5 after the intracranial surgery was performed, rotational behavior was caused by methamphetamine administration (5 mg/kg s.c.) to all groups. Figure 7a depicts the number of ipsilateral rotations recorded during the test, showing that the i.p. pretreatment with Hc-TeTx 0.5 mg/kg reduces the number of ipsilateral rotations caused by the MPP+ treatment. The Hc/MPP+ group had a decreased number of ipsilateral rotations (2.3 ± 0.6 turns/min; mean ± SEM), as compared to the MPP+ group (11.2 ± 2.4 rotations/min). The results show that the Hc/MPP+ treatment decreased the number of turns per minute by approximately 82%, as compared to the group with the MPP+ lesion. The vehicle and Hc-TeTx groups did not show turning behavior by methamphetamine. In the cylinder test, all animals showed, before the surgery, a good performance during the 5 min of vertical exploration (88.5 ± 1.8% in the use of both forelimbs), as shown in

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Ipsilateral striatum

Contralateral striatum

DAT TH

80 kDa 60 kDa

VMAT-2

75 kDa 55 kDa 45 kDa

β-Acn

TH (% of control)

DAT (% of control)

52 kDa

VMAT-2 (% of control)

Fig. 5 Western blot analysis of dopaminergic terminal markers (dopamine transporter, DAT; tyrosine hydroxylase, TH; vesicular monoamine transporter2, VMAT-2; and β-actin) in both ipsilateral and contralateral striata on the fifth day after intrastriatal injection, as indicated in the figure. Total protein extracts from striatal tissue were prepared as stated in the BMaterials and Methods.^ No significant differences were detected in the contralateral side, whereas in the ipsilateral hemisphere, the intrastriatal injection of MPP+ (10 μg) caused a general decrease in the presence of dopaminergic terminal markers (DAT, TH, and VMAT-2) of approximately 50%. Pretreatment with Hc-TeTx markedly preserved the markers’ content. In each case, a representative blot is shown in the upper part and OD quantification of the signals is shown in the lower part. The results are expressed as the mean values ± SEM of four experiments performed separately. n = 3–4 animals per group and experiment. Data were analyzed using ANOVA and post hoc Bonferroni tests (vs control *p < 0.05, **p < 0.01, ***p < 0.001; MPP+ vs Hc/MPP+ # p < 0.05, ##p < 0.01)

Fig. 7b. After surgery, only the MPP+ group had a significant decrease in the use of both forelimbs (17 ± 3%), associated with dopaminergic denervation in the striatum. In contrast, the group pretreated with Hc-TeTx (Hc/MPP+) had a significantly better performance (80 ± 6%), and both the control and the Hc-TeTx groups show a good performance (91 ± 1.4 and 76 ± 2.9%, respectively). The stepping test represents a measure of the skills of the forelimb movement and is a good model for the assessment of akinesia in PD. The values for all animals prior to surgery were similar (16 ± 1 number of steps, n.s.) for the contralateral and ipsilateral paws (Fig. 7c, d). After the lesion with MPP+, the performance with the contralateral paw on day 17 was significantly impaired (1.6 ± 0.9, n.s), with respect to the ipsilateral paw (15.0 ± 0.6, n.s.). In contrast, the Hc/MPP+ group did not present an important impairment of the contralateral paw (14.2 ± 0.6,

n.s.) nor of the ipsilateral paw (15.9 ± 0.4, n.s.), as shown in Fig. 7d. Most importantly, Hc-TeTx prevents the motor impairments throughout the remaining 22 days after the dopaminergic lesion. The control and Hc-TeTx groups showed similar results with contralateral and ipsilateral paws regarding the number of steps (13 ± 1 number of steps), meaning that the motor asymmetry was not affected in these groups (Fig. 7c).

Discussion Our goal in the present study was to mimic a possible early stage of PD, in which no dopaminergic neuron death is detected in the SNpc but dopaminergic impairment in the striatum is found, being in agreement with the conclusions reported by Herkenham et al. (1991) and Dauer and Przedborski

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Contralateral

b

SNpc VTA

c

Hc / MPP+

MPP+

Hc-TeTx

Control

a

Fig. 6 Assessment of the dopaminergic neuron content in the SNpc. The number of positive TH immunoreactive neurons was determined by counting every sixth section covering the entire extent of the SNpc. TH inmunoreactive cells in both ipsilateral and contralateral hemispheres were counted using a ×40 objective. a Representative images of the area including substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) from injected animals (×2 magnification). Scale

bar, 200 μm. b Detail of the TH-positive cell bodies of the SNpc at ×40 magnification. Scale bar, 20 μm. c Quantification of the number of THpositive neurons in SNpc from control and from treated animals, showing no significant differences between groups. The results are expressed as the mean values ± SEM obtained in two separate experiments (each group n = 5). The one-way ANOVA statistical test and Bonferroni post hoc test show no significant differences, p > 0.05

(2003) in a rodent model. This BPD mild model^ was created by infusing intrastriatally a low quantity of MPP+ (approx. 30 nmol), and thereafter, the efficiency of Hc-TeTx to prevent the degeneration of the dopaminergic innervation in the striatum and, consequently, the possibility to retard PD progression, was tested. In the brain, MPP+ enters dopaminergic neurons through the DAT, present both in the dendrites, in the SNpc, and in axons and terminals, in the nucleus striatum (Hersch et al. 1997; Chagkutip et al. 2003). Consequently, intranigral or intrastriatal MPP+ infusion in rat can generate neurotoxicity in dopaminergic neurons. The degree of toxicity depends on the amount of MPP+ injected, usually 60–100 nmol MPP+ being applied, leading to dopaminergic SNpc cell death by apoptosis (Banerjee et al. 2007; Chuang and Chen 2002; Ghorayeb et al. 2002; Lo et al. 2008; Obata et al. 2008; Rubio-Osornio et al. 2009; Yazdani et al. 2006). In our experiments, the intrastriatal infusion of MPP+ leads to a decrease of the striatal DA content of the ipsilateral brain hemisphere on the third day after the stereotaxic injection, with maximal depletion being observed 5 days after the intervention. In contrast, the infusion of 6-OHDA causes great striatal depletion of DA (> 90%) and metabolite long-term effect after

neurotoxin injection, allowing progressive damage on dopaminergic system (Solís et al. 2016, 2017). The moderate diminution of DA and metabolites caused by the infusion of MPP+ was appropriate for our purposes, and therefore, we decided to perform the subsequent experiments at this specific time-point. The DA depletion was in accordance with the observed levels of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis (Nagatsu et al. 1964), which also underwent a decrease of about 50%. The absence of dopaminergic cell death in the SNpc, verified by TH-positive cell counting and by Fluoro-Jade B staining, an anionic fluorescein derivative which specifically labels dead or dying neurons (data not shown), demonstrated that the effect exerted by MPP+ injection did not reach the SNpc at the time of analysis, acting only locally in the site of injection, i.e., the nucleus striatum. In fact, it has been described that injection of MPP+ causes a progressive and irreversible alteration of DAT activity in dopaminergic neurons and a subsequent fall in the number of DAT transporters and, thus, a reduction of DA uptake into the dopaminergic neurons (Chagkutip et al. 2003). The presence of DA in the synaptic space results in site-specific damage to synaptic buttons of dopaminergic nerve terminals due to DA auto-oxidation or DA enzymatic

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Fig. 7 Pretreatment with Hc-TeTx improves the asymmetric behavior in rats with MPP+. The tests were evaluated in rats with previous Hc-TeTx treatment, using a dose of 50 ng/kg i.p., or vehicle followed by dopaminergic lesion caused by injection of MPP+ into the striatum. a The rotations were caused by methamphetamine (5 mg/kg s.c.). The group Hc/MPP+ had significantly less rotations than the MPP+ group. The vehicle and Hc-TeTx groups did not show rotational behavior during the test. b The number of wall contacts performed simultaneously by the ipsilateral and contralateral paws during 5 min inside the cylinder was recorded during 3 weeks, and the graph shows

the percentage of both forelimbs use. The group Hc/MPP+ had an improvement of the percentage of both forelimbs use with respect to the MPP+ group. c Time-course of the performance in the adjusting step test for the control groups, as the graph shows that there are no changes between the Hc-TeTx and control group. Finally, the graph (d) shows that Hc-TeTx improves the number of steps during the test in contrast with the MPP+ group. The data are the mean ± SEM, **p < 0.01, ***p < 0.001, #p < 0.0001 significantly different compared to MPP+ animals, using one-way ANOVA for repeated measures followed by Bonferroni’s multiple comparison test

oxidation and, hence, generation of reactive hydroxyl radical ·OH and other reactive oxygen species (ROS), which are able to initiate membrane lipid peroxidation (Hastings 2009; Lotharius and O’Malley 2000; Barc et al. 2002; Obata 2002). In addition, MPP+ injection impairs the complex I of the mitochondrial electron transport chain, leading to a deficit in ATP formation and an increase of ROS production, i.e., energetic crisis and oxidative stress (Przedborski et al. 2004). The ROS cause a site-specific degeneration of the synaptic buttons of dopaminergic nerve terminals in the nigrostriatal system (Obata et al. 2008), leading to a depletion of dopamine and to a retrograde degeneration of the dopaminergic neurons.

It is clear that MPP+ and 6-OHDA share some effects such as inhibiting electrotransport chain complex I, which induces oxidative stress that results in apoptosis and necrosis which are the result of both neurotoxins. Interestingly, the intrastriatal injection of 6-OHDA causes progressive retrograde neuronal degeneration in the substantia nigra and ventral tegmental complex, and its motor effects progress as well as the neurodegenerative process. Over a brief timecourse (12 h to 2–3 days), neuronal death is found after 6OHDA. Once transported into the neurons, 6-OHDA is oxidized, then the oxidized molecule generates free radicals, inhibits mitochondrial complex I, and produces superoxide

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and hydroxyl radicals (Blum et al. 2001). It is not only toxic to the DAergic neurons but can also induce microglial activation. In contrast, the effects of MPTP on animals depend on several parameters, such as the administration mode, dosage, and animal age. The MPP+ model is useful to show the brief dopamine deficit after its intrastriatal injection. Also, its behavioral consequences can be explored, but the system is able to recover after some time and a new administration of the Hc-TeTx fragment will be necessary in order to maintain the low dopamine levels in the rat brain. Moreover, we could examine the neuroprotective effects of Hc-TeTx fragment in each toxic environment, since both represent an interesting tool to model nigral degeneration, and lead us to explore different motor deficits in rats. Thus, all of these studies are necessary to confirm the potential therapeutic efficacy by the Hc fragment. Then, the very first events to take place in PD, even before no symptoms are observed, would take place in the dopaminergic terminals located in the nucleus striatum and would be retrogradely transmitted to the cell body located in the SNpc, thus fitting with the Bdying back^ model proposed by some authors with the name of dysferopathy (Serulle et al. 2007). Thus, the low amount of MPP+ infused in the striatum causes terminal degeneration but does not induce the Bdying back^ effect, i.e., the loss of neuronal bodies in the SNpc, at the time of analysis. Therefore, we observed TH-fiber recovery on day 7 postinjection of MPP+, and these results are consistent with other experiments (Espino et al. 1994, 1995) and with other neurotoxins such as METH. Its neurotoxicity mechanisms are not totally distinct to MPP+, thus involving oxidative stress, neuroinflammation, and proapoptosis as was observed by Ares-Santos et al. (2014); 3 days after METH application, TH-fiber recovery occurs, and although there were TH/Ag cell bodies which were positive in SNpc since 12 h after METH, on day 7, the neurons in the neurodegeneration process decreased, therefore allowing the TH-fiber recovery in the striatum. Also, in PD and in neurotoxin animal models (6OHDA, MPTP), the depletion of striatal DA leads to alterations in glutamatergic neurotransmission (Solís et al. 2016; Rodrigues et al. 2007), which manifested as a reduction in dendritic arborization and spine density (Suárez et al. 2014, 2016; Toy et al. 2014; Villalba and Smith 2010). Similar findings have been made using MPP+ in triple organotypic-slice cultures largely recapitulated in the in vivo connections between the substantia nigra, striatum, and cortex from rats, finding that thin spines were preferentially lost after dopamine depletion caused by MPP+ (Neely et al. 2007). Therefore, in subsequent studies, it would be interesting to evaluate how the instrastriatal infusion of MPP + in rat modifies the corticostriatal plasticity.

Therefore, in the present study, an animal model is provided that mimics a possible initial stage of PD and a therapy based on the peripheral injection of the recombinant Hc-TeTx fragment from the tetanus toxin. Hc-TeTx lacks the toxicity of the whole toxin and possesses the ability to bind to terminal neurons and of being retroaxonally transported (Deinhardt and Schiavo 2005; Deinhardt et al. 2006; Schellingerhout et al. 2009). In our model, the application of Hc-TeTx as a pretreatment prior to the MPP+ intracranial injection was able to prevent the DA and metabolites to fall produced by MPP+. According to this result, the tyrosine hydroxylase levels, as well as those of the DAT and VMAT-2 dopaminergic markers, were partially restored. By studies of radioligand uptake, we confirmed that HcTeTx fragment does not interfere with dopamine or MPP+ transport, with the kinetic parameters remaining unchanged at Hc-TeTx doses that we consider therapeutic. This demonstration is important because tetanus toxin, through its carboxyl-terminal domain, specifically inhibits the uptake of serotonin, an aminergic system that shares transporters with catecholamine systems, with high homology and functionality (Inserte et al. 1999; Najib et al. 1999). However, despite the structural and functional similarity, the Hc-TeTx fragment inhibits only the transport of serotonin but not of DA. Furthermore, we have demonstrated that the Hc-TeTx fragment has the ability to avoid the striatal dopaminergic degeneration triggered by the oxidative stress produced by MPP+. One feasible explanation for the Hc-TeTx action is the maintaining of the cells’ natural equilibrium between oxidants and antioxidants by regulating the expression of genes related to ROS detoxification that triggered Nrf2 activation and nuclear translocation, where Nrf2 binds in antioxidant response element (ARE) to regulate antioxidant genes, such as SOD, catalase, or glutathione peroxidase. Also, dopaminergic neurons are particularly vulnerable when Nrf2 fails as a key regulator of redox (Granado et al. 2011); some neurotrophins such as BDNF in vivo induce nuclear translocation of Nrf2. Although the mechanism is not clear yet (Bouvier et al. 2017), several studies show that Nrf2 nuclear translocation is dependent on MAPK activation. Therefore, if the Hc-TeTx fragment activates Akt and ERK1/2 pathways (Gil et al. 2003), this could induce the activation of Nrf2 and provide an antioxidant environment to protect from MPP+ neurotoxicity. What is perhaps more relevant than biochemical data are our results found in motor behavior tests which show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP+-treated rats. In vivo approaches have also demonstrated that co-application of Hc-TeTx and MPP+ in the ipsilateral striatum is neurorestorative in the same partial lesion model of Parkinson’s disease used in the present work, thus maintaining the DA and related

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metabolite levels in the striatum and ameliorating motor behavior (Mendieta et al. 2009). It is noteworthy that intraperitoneal application of extremely low doses of the HcTeTx fragment yields levels of protection, in terms of catecholamine restoration and of motor behavior, similar to those observed with intrastriatal Hc-TeTx application in the same model (Mendieta et al. 2009). As we have indicated, the Hc-TeTx fragment induces similar protective mechanisms in MPP+-induced PD model as those induced by growth factors such as GDNF or mesencephalic, astrocyte-derived neurotrophic factor (MANF) (Voutilainen et al. 2009). These and other neurotrophic factors showed robust protective and reparative activities in various animal models of neurodegenerative diseases but were unsuccessful in diffusing significant distances (Hamilton et al. 2001; Salvatore et al. 2006), probably due to the poor transport of these factors in the CNS. Thus, in order to progress in the study of the therapeutic use of the Hc-TeTx recombinant molecule, our group proposes a breakthrough, injecting the Hc-TeTx fragment peripherally, instead of intracranially, in the described neurodegeneration animal models as in previous studies we have indicated (Mendieta et al. 2012; Sánchez-González et al. 2014; Mendieta et al. 2016; Palafox-Sánchez et al. 2016). The rationale behind this i.p. administration is based on the singular characteristics of the Hc-TeTx fragment, which are its specific binding and uptake into neuromuscular and sensory nerve terminals and its capacity to experience retroaxonal transport and trans-synaptic jumps, thus gaining access to almost all of the CNS (Kissa et al. 2002; Binz and Rummel 2009; Francis et al. 2004; Roux et al. 2005). This application has the obvious advantage of being less invasive than intracranial application, which has been proposed for classical trophic factors used so far (Love et al. 2005). It is remarkable that immunization does not interfere with the uptake and transport by motor neurons of Hc-TeTx in mice (Fishman et al. 2006). Advances in brain imaging technologies and the identification of genetic or biochemical markers for this disease should lead to earlier diagnosis and provide the opportunity for therapeutic intervention with the Hc-TeTx fragment at an early stage, when a significant number of the dopaminergic neurons remain healthy. In summary, pretreatment with the Hc-TeTx fragment in hemiparkinsonian rats prevents the decrease of dopamine as well as other dopaminergic markers such as TH, DAT, and VMAT-2 in the striatum due to injury with MPP+, and also, we observed improvement of motor behaviors. These results point to the potential effectiveness of Hc-TeTx fragment as a therapeutic agent in this Parkinson’s disease model and, together with previous results, open the possibility to its use in dysfunctions that induce neuronal death, such as ischemia, amyotrophic lateral sclerosis, or Alzheimer’s disease.

Acknowledgements We thank Chuck Simmons for his help in the English preparation of this manuscript. Funding Information This work was supported by Grants SAF201343900-R and SAF2016-80027-R from the Ministerio de Ciencia e Innovación (Dirección General de Investigación) of the Spanish Government. L. Mendieta received a grant from PRODEP-SEP (PTC472) and V. Palafox-Sánchez received a scholarship from CONACYTMexico (244867).

Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflicts of interest. Abbreviations DA, dopamine; DAT, dopamine transporter; DOPAC, 3,4-dihydroxyphenylacetic acid; Hc-TeTx or Hc, carboxyl-terminal domain of the heavy-chain of tetanus toxin; HVA, homovanillic acid; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; 3 MT, 3-methoxytyramine; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter-2

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