Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, caused by selective degeneration of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc) [1, 2]. Dopaminergic deficit results in motor disabilities, such as rest tremor, bradykinesia, rigidity of movement, and postural instability [3]. Despite the existence of specific genetic causes, PD is primarily a sporadic disorder. Although the etiology of PD remains unknown, environmental or genetic factors might contribute to the pathogenesis of PD, leading to an increase of oxidative stress, endoplasmic reticulum (ER) stress, and impairing mitochondrial function [4–7]. Besides dopaminergic neuronal death, neuropathological hallmarks of PD include glial reactivity, inflammation, and intracellular proteinaceous inclusions called Lewy bodies (LB), which are mainly constituted by α-synuclein aggregates [4, 6].

Genetic and transgenic animal models support the idea that the accumulation of fibrillar α-synuclein plays a relevant role in the pathogenesis of PD [8]. However, mounting evidence [9] indicates that soluble pre-aggregate oligomers are responsible for neurodegeneration, having aggregates a clearance function since they could immobilize the oligomeric α-synuclein, avoiding its toxic availability.

Abnormal forms of α-synuclein and oligomers lead to cellular homeostasis imbalance that includes alterations of calcium signaling [10, 11], mitochondrial dysfunction [12], oxidative and nitrosative damage [13], changes in cytoskeletal integrity [14], and neuroinflammation [15].

Despite the ubiquitous presence of α-synuclein in LB [16] and its relevance in the development of PD [8], the molecular mechanism mediating the neurotoxicity of α-synuclein remains unclear and still in debate [17].

Cytosolic and membrane bound levels of α-synuclein are regulated by their synthesis, degradation, and secretion rates and a proteostasis imbalance has been shown to be involved in aggregates formation. The ubiquitin-proteasomal system dysfunction contributes to inclusions formation and toxicity [18, 19]. Furthermore, post-translationally modified α-synuclein is impaired to be degraded by the autophagy-lysosome pathway [20], indicating that disturbance of the two main routes responsible for protein degradation triggers α-synuclein accumulation in the cell, leading to ER stress.

Approximately, one third of cellular proteins are synthesized in the ER where they are folded and undergo post-translational modifications, and transit to membrane compartments or are secreted [21]. However, under certain conditions of cellular stress, protein folding is altered, and misfolded substrates accumulate in the lumen of ER triggering an adaptive signaling pathway called unfolded protein response (UPR) [21, 22]. This mechanism is regulated by the ER resident chaperone glucose-regulated protein-78 (GRP78) that mediates the activation of three major stress sensors: protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme-1 (IRE1), and activating transcription factor-6 (ATF6) [21]. The initial role of UPR is to restore homeostasis and alleviate ER stress through attenuation of protein translation, increase folding capacity, ER membrane expansion and activation of ER-associated degradation (ERAD pathway) [21–24].

Endoplasmic reticulum (ER) stress has recently acquired great attention in the field of neurodegenerative diseases [25, 26], particularly with respect to PD [27]. In fact, post-mortem analysis has demonstrated that substantia nigra of PD cases present activation of the PERK pathway, which is correlated with increased levels of α-synuclein in dopaminergic neurons [28]. Additionally, the C/EBP-homologous protein (CHOP), a downstream member of the PERK pathway [21], is up-regulated in the 6-OHDA neurotoxin-induced cell death both in vitro [29] and in vivo [30], corroborating the findings that CHOP knockout mice dopaminergic neurons are protected from cell death after 6-OHDA challenge [31]. Besides PERK, the IRE1 and ATF6 branches of the UPR have also been implicated in PD [27], however most of the experimental data were obtained employing cultured cell lines or neurons [32, 33] and transgenic animals [34, 35]. These data collectively suggest that the UPR is activated in PD and that UPR activation is closely associated with the accumulation and aggregation of α-synuclein and dopaminergic cell death.

Because increasing evidences point to the pivotal role the ER stress and UPR in PD and there is lacking of in vivo ER stress models that use only non-genetically manipulated animals, we developed a new ER stress rodent model employing an intranigral injection of the well-known ER stressor tunicamycin (Tm) [36]. First, we found that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with control. In this context, the classical 6-OHDA neurotoxin-based model was employed as an established positive control for PD [31, 37, 38]. Finally, animals challenged with Tm presented increased levels of ER stress markers, indicating that the UPR signaling was activated in this new animal model, which in turn recapitulates several features of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.

Materials and Methods

Animals

Experimental procedures were performed in accordance with the Ethics Committee for Research on Animals UFPR (protocol #778). Fifty-eight adult male Wistar rats 9 weeks old, weighing 280–320 g at the beginning of the experiments were used. Animals were housed in groups of five, in polypropylene cages and maintained under standard conditions of temperature (22 ± 2 °C) and illumination (12/12 h light/dark cycle). The animals had free access to water and food throughout the experiments.

Surgical Procedures

Animals were anesthetized using ketamine/xilazine (100 mg/kg; 20 mg/kg; Syntec do Brasil Ltda, Brazil) intraperitoneally, and placed in a stereotaxic frame. The following coordinates were used to the bilateral injury, bregma as a reference: SNpc (AP) =−5.3 mm, (ML) =± 2.1 mm e (DV) =−7.8 mm (Paxinos and Watson, 2005). 5 mg/ml stock solution of Tm (Sigma) was prepared in dimethylsulfoxide (DMSO) (Sigma). Needles were guided to the region of interest for a bilateral infusion of 0.5 μL of Tm prepared at a final concentration of 0.2 mg/mL diluted in PBS (1:25 dilution from the stock solution). As a positive control of the lesion, 1 μL of the neurotoxin 6-hydroxydopamine (6-OHDA) (Sigma) dissolved in PBS at a final concentration of 1 mg/mL, supplemented with 0.2 % of ascorbic acid was injected. Also, the sham procedure (control group) received 1 μL of the vehicle (PBS:DMSO, 1:25). The infusions were performed with an electronic pump (Harvard Apparatus, Holliston, MA, USA) at a flow rate of 0.33 μL/min. After each infusion the needle was kept in the injection site for additional 2 min to avoid reflux.

Footprint Analysis

One week after surgery, animals were evaluated for motor coordination and balance [39]. A 100-cm long, 10-cm wide runway (with 35-cm high walls) was used for footprint test. Before collecting footprints, each animal was able to explore the runway for 5 min. After recognition, animal’s paws were painted with water-based nontoxic paint (using different color for hindpaws and forepaws), and each animal was immediately placed at one end of the runway previously covered with a strip of paper as template. Data were collected when the animal was able to walk without interruption giving at least four steps in a straight line for each paw (initiation and finishing of the run were ignored) [40, 41]. The paper was removed for analysis and fore and hind step parameters were analyzed according to an established methodology [40].

Western Blot Analysis

To determine ER stress within the substantia nigra (SN), 7 days after injection with 6-OHDA or Tm, animals were decapitated, their brains were rapidly ice-removed and SN was dissected. Until processed for analysis, tissues were stored at −80 °C in lysis buffer containing 50 mM NaCl, 50 mM Hepes, 2 mM EDTA, 1 % Triton X-100, 1 mM PMSF, 1 mM sodium orthovanadate, and complete protease inhibitor mixture EDTA-free (Roche). Samples were manually homogenized on ice using a micropestle (Eppendorf). After centrifugation (10 min 12,000×g at 4 °C), supernatant was collected. Protein concentration was determined by the Bradford method (Bio-Rad, Germany). Samples were subjected to SDS-PAGE and proteins transferred to a nitrocellulose membrane (GE Healthcare). The membranes were then blocked in 5 % slime milk or BSA in TBS-T (blocking solution) for 1 h at room temperature and incubated 16 h with the desired antibody diluted in blocking solution. Employed antibodies were: mouse monoclonal anti-β-actin (A5441, Sigma), anti-α-synuclein (610787 BD), anti-CHOP (MA1-250, Thermo Scientific), anti-KDEL (ADI-SPA-827, Enzo Life Science) and anti-GAPDH (#2118, Cell Signaling). After primary antibody incubation, membranes were extensively washed with TBS-T and incubated with HRP-conjugated secondary antibody (Sigma) in blocking solution for 1 h at room temperature. Finally, membranes were washed again and immune complexes were detected using the ECL chemiluminescent detection system (GE Healthcare Life Sciences, Brazil). The protein levels were quantified by densitometry using ImageJ v1.47 software (National Institutes of Health, USA).

Imunohistochemistry

Animals were deeply anesthetized using ketamine/xilazine (100 mg/kg/20 mg/kg), and transcardially perfused with PBS, followed by 4 % paraformaldehyde in PBS (pH 7.2) at a constant flow of 1 mL/min. Fixed brains were quickly removed and post-fixed by immersion in 4 % paraformaldehyde for 24 h. The brains were immersed in 30 % sucrose solution for cryoprotection, and stored at −80 °C until cryostat sectioning (Leica Lm 1850, Germany). Brains were sliced into 30-μm coronal sections (Bregma: −5.16 to −5.40) and stored in anti freezing solution (30 % ethylene glycol, 30 % sucrose in PBS) at −20 °C. Free-floating sections were permeabilized for 15 min at room temperature with 0.1 % Triton X-100 in PBS, treated with 0.3 % H2O2 for 20 min, washed three times with PBS followed by blocking with 5 % BSA for 30 min and incubation overnight with mouse anti-tyrosine hydroxylase (TH) (T2928, Sigma) or rabbit anti-GFAP (Z0334, DAKO) antibody at 4 °C. Antibodies were detected using HRP immunostaining kit (Diagnostic Biosystems). Tissues were dehydrated in aqueous graded ethanol solutions from 70 to 100 % and then immersed twice in xylene and mounted with Entellan New (Merck).

Statistical Analysis

Differences between the groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey post hoc tests. Values are expressed as mean ± standard error of mean (SEM). The level of significance was set at P ≤ 0.05.

Results

We first examined whether the ER stressor Tm could induce locomotor disturbances in animals subjected to direct drug delivery into SNpc, as typically observed in other well-established animal model of PD [31, 37, 38]. Seven days after challenging rats with a single bilateral injection of Tm or 6-OHDA, footprint test was performed to assess locomotor performance. Quantitative analyzes showed absence of statistical differences in the stride length of hindpaw and forepaw between groups (Fig. 1a). While the stance width of the forepaws did not show difference between groups, hind paws instead was significantly broader in Tm- and 6-OHDA-injected (P < 0.01) animals [F [2, 24]= 7.986; P = 0.0022] (Fig. 1b) in comparison with the control group.

Fig. 1
figure 1

Footprints reveal gait abnormalities in animals challenged by Tm. a Stride lengths and b stride width for hind- and fore-paws measured with the footprint test. Data is represented as the mean ± SEM (CONTROL n = 10; TUNICAMYCIN n = 8; 6-OHDA n = 9), asterisks indicate statistical differences (***) p ≤ 0.01 compared with the control group

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After locomotor assessment, animals were sacrificed and brains evaluated for dopaminergic neuronal loss by TH immunostaining at SNpc level. Figure 2 shows representative images of coronal midbrain sections of TH-immunoreactive neurons for control (sham), Tm and 6-OHDA (positive control) groups (Fig. 2a–c, respectively). Quantification (Fig. 2d) of TH-immunopositive neurons in SNpc showed a decrease of 25 % (P < 0.05) and 22 % (P < 0.05) for Tm and 6-OHDA groups respectively, when compared to the control group [F [2, 10]= 6.497; P = 0.0156].

Fig. 2
figure 2

Dopaminergic cell loss induced by Tm. Representative microphotographs showing TH-immunoreactivity of midbrain coronal sections of a Control, b Tm and c 6-OHDA injected animals 7 days post-lesion. d Quantification of TH-positive cell number in SNpc. Data are represented as the mean ± SEM (CONTROL n = 5; TUNICAMYCIN n = 4; 6-OHDA n = 4). Asterisks indicate statistical differences (**) p ≤ 0.05

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As a hallmark for neurodegeneration, reactive astrogliosis was examined with antibody against GFAP (Fig. 3) [42]. GFAP immunohistochemistry of coronal midbrain section from Tm and 6-OHDA injected animals showed ~2.6 (P < 0.0001) and ~2.9 increasing fold (P < 0.0001) of GFAP+ astrocytes when compared to control animals [F [2, 10]= 33.11; P < 0.0001], respectively (Fig. 3d). In addition, astrocytes from Tm and 6-OHDA animals presented morphological changes [42] such as cellular hypertrophy and up-regulated expression of GFAP (Fig. 3b’–c’, respectively), when compared to control animals (Fig. 3a’).

Fig. 3
figure 3

Reactive astrogliosis in response to Tm lesion. Representative microphotographs showing GFAP-immunoreactivity in SN from a Control, b Tm and c 6-OHDA injected animals 7 days post-lesion. (a’–c’) Higher magnifications of the boxed areas in a–c, showing morphology of GFAP-positive cells for Control, Tm and 6-OHDA groups, respectively. d Quantification of GFAP-positive cell number in SNpc. Data are represented as the mean ± SEM (CONTROL n = 5; TUNICAMYCIN n = 4; 6-OHDA n = 4). Asterisks indicate statistical differences (****) p ≤ 0.0001

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Based on previous studies that associate ER stress to protein misfolding [43–45], and because α-synuclein misfolding is a hallmark of PD [28, 46], we analyzed α-synuclein expression and its oligomeric status by western blot after Tm treatment. Interestingly, analysis of α-synuclein expression showed no difference in monomeric α-synuclein levels between groups (P = 0.4336) (Fig. 4a, mid panel and Fig. 4b). However, Tm injected animals showed increased high molecular weight forms of α-synuclein (P < 0.01) [F [2, 11]= 9.633; P = 0.0038] (Fig. 4a, upper panel and Fig. 4c) indicating that the ER stressor Tm can induce in vivo one of the molecular features of PD.

Fig. 4
figure 4

Tunicamycin induces α-synuclein oligomerization. a Tissue homogenates from SN were immunoblotted against α-synuclein and GAPDH for Control, Tm and 6-OHDA 7 days after treatment. b Immunoblot quantification of monomeric α-synuclein and c oligomeric α-synuclein. Data are represented as the mean ± SEM (CONTROL n = 5; TUNICAMYCIN n = 4; 6-OHDA n = 5). Asterisks indicate statistical differences (***) p ≤ 0.01

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As a major consequence of ER stress, UPR activation triggers repression of translation and up-regulation of ER-resident chaperones after accumulation of misfolded proteins [47, 48]. In order to measure the intensity of ER stress observed after injury, ER-resident chaperones were evaluated by western blot with anti-KDEL tetrapeptide signal (Fig. 5a). Quantification of ER-resident chaperone levels presented an ~2.9 and ~2.5-fold increase for GRP78 (P < 0.05) [F [2, 12]= 6.411; P = 0.0128] (Fig. 5c), and ~9.4 (P < 0.001) and ~7.5-fold (P < 0.05) increase for GRP94 for Tm- and 6-OHDA-treated animals, respectively [F [2, 12]= 8.695; P = 0.0046], when compared to control animals (Fig. 5b), indicating that both Tm and 6-OHDA challenges increase ER-resident chaperones.

Fig. 5
figure 5

Tunicamycin and 6-OHDA induce ER-stress in SN. a Tissue homogenates from SN were immunoblotted against KDEL (GRP78 and GRP94) and β-actin for control, Tm and 6-OHDA 7 days after treatment. b–c Quantification of GRP78 and GRP94 are represented as the mean ± SEM (CONTROL n = 5; TUNICAMYCIN n = 5; 6-OHDA n = 5). Asterisks indicate statistical differences (**) p ≤ 0.05, (***) p ≤ 0.01

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Upon ER stress, GRP78/GRP94 luminal chaperones dissociate from ER-resident membrane proteins, which transmit downstream UPR activation signals to the cytoplasm [21, 49]. To clarify if the dopaminergic lesion (Fig. 2) was accompanied by PERK pathway signaling modulation, we performed western blot analysis of CHOP in SN samples (Fig. 6a). Despite the PERK-CHOP pathway has been described as the major UPR-associated pro-apoptotic branch [21, 50] we only observed a tendency to increase levels of CHOP in Tm group (Fig. 6b, P < 0.10) when compared to the control group, suggesting that other UPR pathway could be involved in neuronal cell death. Confirming previously described results [30], animals presented increased expression of CHOP under 6-OHDA treatment (Fig. 6b, P < 0.05) [F [2, 15]= 4.649; P = 0.0268], reinforcing the role of ER stress in these established animal PD model.

Fig. 6
figure 6

Activation level of the pro-apoptotic PERK pathway after Tm and 6-OHDA injections. a Tissue homogenates from SN were immunoblotted against CHOP and β-actin for protein loading control. b Quantification of CHOP is represented as the mean ± SEM (CONTROL n = 6; TUNICAMYCIN n = 6; 6-OHDA n = 6), asterisks indicate statistical differences (**) p ≤ 0.05

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Discussion

There are several factors that may be associated with the genesis of neurodegenerative diseases. Genetic causes, inflammation, ATP generation deficiency, increases in ROS production, mitochondrial dysfunction, ER stress and protein degradation pathways have been associated with neurodegenerative progression in PD both in animal models and humans [25, 51]. In the present study, we propose a novel in vivo model to the study of Parkinsonism induced by ER stress.

Increased expression of ER-resident chaperones, such as GRP78 and GRP94 is thought to be a response against ER stress, in order to increase protein-folding capacity and alleviate stress conditions (21, 48, 49). In view of that, previous in vitro studies demonstrated that 6-OHDA, 1-methyl-4-phenylpyridinium (MPP+) and rotenone induce activation of ER-stress pathways and promote cellular death in neuronal cultures [29, 32], indicating that known drugs that induce PD also induce ER stress. 6-OHDA is a classical neurotoxin that leads to PD by mechanisms of selective dopaminergic neuronal death by H2O2 and quinone generation [52]. The fact that cultured sympathetic neurons from PERK-/- knockout mice were more susceptible to die than wild-type neurons when exposed to 6-OHDA indicates the importance of ER stress in PD pathophysiology [32] and suggests that other UPR pathways may contribute to induce cell death. Moreover, mice with 6-OHDA intracerebroventricularly injected showed GRP78 increase in striatum [30]. Our data extend these findings as demonstrated by the increase in both GRP78 and GPR94 expression levels after 6-OHDA treatment and reinforce the role of the ER stress in the 6-OHDA neurotoxicity rat model. In addition, the ER stressor employed in this study, Tm, also increased levels of GRP78 and GRP94 7 days after lesion (Fig. 5a–c), as similarly observed for GRP78 after ventricular administration of Tm and thapsigargin [53]. Although CHOP induction is classically associated to PERK-eIF2α-ATF4 pathway, CHOP levels of Tm injected animals showed no statistical difference when compared to the control group (Fig. 6), suggesting that increased levels of GRP78/GRP94 after lesion (Fig. 5a–c) could be related to other ER-stress pathways, like ATF6 or IRE1 [54–57]. Taken together, these results suggest that ER stress is a common event in these models of PD.

Under ER stress conditions, activation of PERK pathway leads to eIF2α phosphorylation, activating transcription factor 4 (ATF4), CHOP and apoptosis [21, 50]. Different studies demonstrated, in vivo and in vitro, that CHOP-knockout cells are less susceptible to ER stress induced apoptosis [58–60]. This was confirmed later by CHOP knockdown in neuronal cultures that present increased neuronal survival after Tm or thapsigargin exposure [61]. In fact, chop-/- mice show protection signs after injection of 6-OHDA in SNpc of adult mice [31], and wild-type mice intracerebroventricularly injected with the same neurotoxin showed an increase in CHOP expression (30). Surprisingly, Tm injected animals did not exhibit increased levels of CHOP when compared to 6-OHDA (Fig. 6), suggesting that neuronal loss induced by Tm injection could be associated to other apoptotic pathways, different from CHOP.

In PD, protein misfolding and aggregation have become one of the central focuses of research. Accumulation of α-synuclein has been linked to progression of neurodegeneration in PD [62, 63], and different studies had related α-synuclein oligomerization with ER-stress condition. Employing an A53T αS transgenic mice model, Colla and collaborators showed that symptomatic animals presented increased levels of ER-chaperones and α-synuclein ER accumulation in addition to the α-synuclein oligomers formation and disease progression [45]. Remarkably, in vitro studies showed that increased levels of ER stress induce α-synuclein oligomerization in cells overexpressing wild-type α-synuclein [44, 64]. In the current study, we employed an in vivo pharmacological model of ER stress and showed that the endogenous α-synuclein is extensively oligomerized (Fig. 4a, c) under a Tm challenge. Importantly, animals used in the experiments do not have any genetic manipulation (e.g., α-synuclein transgenic mice), indicating that endogenous levels of α-synuclein are sufficient to produce oligomerized forms of the protein under ER-stress conditions. Interestingly, in accordance with previous results obtained with human neuroblastoma cell line and mice primary neuronal cultures [44, 64], monomeric α-synuclein levels did not change after Tm treatment (Fig. 4a, b), suggesting that oligomerization is not caused by an increased α-synuclein synthesis. In the present study, we observed that Tm treatment was more efficient to produce oligomerized forms of α-synuclein, when compared to 6-OHDA-rat model (Fig. 4a, c). The observed difference in such oligomerization status between Tm- and 6-OHDA- injected animals could be a consequence of the activation of different UPR-associated pathways due to the nature of each ER stressor molecule. However, our results strongly suggest that not only Tm treatment produces α-synuclein oligomers but also 6-OHDA. Unexpectedly, the status of α-synuclein oligomerization has not been previously reported for animals administrated with 6-OHDA, in spite of being a well-established model of Parkinsonism. Further in vivo experiments will be conducted in order to clarify the differences between Tm- and 6-OHDA-induced ER stress and to establish 6-OHDA as a bona fide ER-stressor molecule.

Astrocytes are important players in supporting neurons homeostasis [42, 65]. However, glial reactivity is a major hallmark of every neurodegenerative disease, and because of its relationship with neurons, glial cells could promote the start and progression of neurodegeneration [65–67]. When compared to other CNS regions, SNpc presents the lowest amount of astrocytes [65]. Nevertheless, animal models and post-mortem analyses of patients with PD show increased levels of reactive astroglia in SNpc [68, 69], suggesting that when stressed, these astrocytes could contribute to neurodegenerative progress in PD [65]. In the present study we observed an increase in GFAP+ cell number in SNpc of animals treated with Tm and 6-OHDA, suggesting that ER stress could act as an inducer of astroglial reactivity. Reactive astrogliosis has previously been linked to ER stress induction in mice cortex by a liquid nitrogen lesion model [70], Authors observed that penumbra area was predominantly dominated by reactive GFAP+ cells which express high amounts of BiP and OASIS (Old Astrocyte Specific Induced Substance), two proteins involved with UPR signaling [70].

Tm and 6-OHDA lesions inflicted an increase of stance width for hindpaws (Fig. 1b), suggesting that the occurence of a gait imbalance, since they presented respectively a 25 and 22 % reduction of dopaminergic neurons in SNpc (Fig. 2d). This data are in accordance with previous findings showing that after an intranigral injection of 6-OHDA in an hemiparkinsonian model of PD, footprint tasked animals presented increased distance between hindpaws as soon as 4 days after the lesion [38]. To our knowledge, this is the first demonstration that Tm was employed as an ER-stressor directly in SNpc. It was previously demonstrated that when medaka fish was injected with Tm into cerebrospinal fluid cavity animals presented movement disorder, a behavioral sign related to PD [71]. However, this model presents the limitation of inducing an overall ER-stress, including anatomical regions not directly involved with PD. Furthermore, Fouillet et al. (2012) showed that a chronic intraperitoneal administration of Tm, at low doses, produces a preconditioning with mild UPR that generates neuroprotection in the 6-OHDA mouse model of PD [72]. In contrast, our proposed model employed intranigral drug administration by stereotaxic surgery, which has the advantage to ensure an acute challenge only in a limited region of the CNS and precludes an undesired systemic drug delivery.

Overall, our data confirm that Tm induces in vivo functional and molecular PD conditions, similarly to those induced by 6-OHDA. Induction of UPR and ER stress led to several features of PD, such as α-synuclein oligomerization, dopaminergic neurons death, and locomotor disabilities. Understanding the genesis of PD is necessary for early diagnosis and advances towards the development of new preventive therapies. We believe that future studies employing Tm lesion models and manipulation of UPR pathways will further characterize the role of the ER stress in the development of PD.