Reduced TH expression and α-synuclein accumulation contribute towards nigrostriatal dysfunction in experimental hepatic encephalopathy

Abstract

Purpose:

The present work examines α-synuclein expression in the nigrostriatal system of a rat chronic hepatic encephalopathy model induced by portacaval anastomosis (PCA). There is evidence that dopaminergic dysfunction in disease conditions is strongly associated with such expression. Possible relationships among dopaminergic neurons, astroglial cells and α-synuclein expression were sought.

Methods:

Brain tissue samples from rats at 1 and 6 months post-PCA, and controls, were analysed immunohistochemically using antibodies against tyrosine hydroxylase (TH), α-synuclein, glial fibrillary acidic protein (GFAP) and ubiquitin (Ub).

Results:

In the control rats, TH immunoreactivity was detected in the neuronal cell bodies and processes in the substantia nigra pars compacta (SNc). A dense TH-positive network of neurons was also seen in the striatum. In the PCA-exposed rats, however, a reduction in TH-positive neurons was seen at both 1 and 6 months in the SNc, as well as a reduction in TH-positive fibres in the striatum. This was coincident with the appearance of α-synuclein-immunoreactive neurons in the SNc; some of the TH-positive neurons also showed α-synuclein immunoreactivity. In addition, α-synuclein accumulation was seen in the SNc and striatum at both 1 and 6 months post-PCA, whereas α-synuclein was only mildly expressed in the nigrostriatal pathway of the controls. Astrogliosis was also seen following PCA, as revealed by increased GFAP expression from 1 month to 6 months post-PCA in both the SN and striatum. The astroglial activation level in the SN paralleled the reduced neuronal expression of TH throughout PCA exposure.

Conclusion:

α-synuclein accumulation following PCA may induce dopaminergic dysfunction via the downregulation of TH, as well as astroglial activation.

Keywords

α-synuclein GFAP hepatic encephalopathy hyperammonaemia substantia nigra tyrosine hydroxylase

Abbreviations

DLB

dementia with Lewy bodies

hepatic encephalopathy

MPTP

1-metil-4-fenil-1,2,3,6-tetrahidropiridina

MSA

multiple system atrophy

6-OHDA

6-hydroxydopamine

Parkinson’s disease

ubiquitin

1 Introduction

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome resulting from acute or chronic liver dysfunction. In humans with HE, it has been suggested that the basal ganglia, which are often associated with disorders of movement (Weissenborn & Kolbe, 1998; Spahr et al., 2000; Layrargues, 2001; Burkhard et al., 2003), may be selectively affected (Spahr et al., 2002; Lin et al., 2012). Chronic hyperammonaemia in the rat resulting from portacaval anastomosis (PCA) is accompanied by neurological symptoms resembling those described for chronic HE in humans (review in Butterworth et al., 2009). Rats exposed to PCA also show motor deficits (Martin, 1986; Steindl et al., 1996), which could be associated with degeneration and/or dysfunction of the dopaminergic system.

Dysfunctional dopaminergic neurotransmission between the substantia nigra pars compacta (SNc) and the dorsal striatum (the nigrostriatal pathway) causes several prominent movement disorders in Parkinson’s disease (PD) (Galvin, 2006). These symptoms may be attributable to a reduction in the production of tyrosine hydroxylase (TH), the rate-limiting enzyme involved in dopamine biosynthesis. The loss of SNc neurons and the presence of Lewy bodies (LBs), composed mainly of α-synuclein (Spillantini et al., 1997; Wakabayashi et al., 2007), are hallmark signs of PD.

α-synuclein, a normally soluble neuronal protein localized to synaptic terminals, is an important regulator in dopaminergic transmission. However, it is well known to turn into aggregates or insoluble fibrils (Spillantini et al., 1997; Giasson et al., 1999; Kramer & Schulz-Schaeffer, 2007), and abnormal aggregation is involved in the pathogenesis of neurodegenerative diseases (Dawson & Dawson, 2003; Ma et al., 2003; Tofaris & Spillantini, 2005). Once aggregation has begun, the normal physiological functions regulated by this protein may be severely compromised (Shidu et al., 2004).

In the nigrostriatal system, the overexpression of α-synuclein inhibits the activity of TH, dopamine synthesis in vitro (Perez et al., 2002; Peng et al., 2005) and in vivo (Lou et al., 2010), and causes the impairment of dopamine release (Pelkonen et al., 2010). α-synuclein overexpression may also cause mitochondrial defects in dopaminergic neurons (Zhu et al., 2011). Indeed, studies on transgenic animals and gene-transfected dopaminergic cells have shown that its overexpression leads to the formation of α-synuclein-positive intracellular inclusions and the degeneration or death of dopaminergic neurons (Masliah et al., 2000; Lo Bianco et al., 2002; Zhou et al., 2002; Yamada et al., 2004). More recently, it has been suggested that, apart from α-synuclein aggregation, increased levels of the protein may be sufficient to trigger neurodegenerative processes (Ulusoy & Di Monte, 2013).

Studies in vitro have demonstrated that α-synuclein treatment can directly cause GFAP reactivity in human astrocytes (Koob et al., 2010). Experimental in vivo studies with α-synuclein mutant mice have shown that the number of GFAP-positive astrocytes is increased in the brainstem of α-synuclein mutant mice (Gu et al., 2010). Astrocytes contain α-synuclein in pathological situations (Wakabayashi et al., 2000), but are not known to synthesize it under normal physiological conditions. It has therefore been suggested that it is released from neurons into the extracellular space (Lee et al., 2005) where it accumulates – as seen in the brains of patients with PD (Lee, 2008). The altered α-synuclein molecule can be captured by the astrocytes (Braak et al., 2007; Lee et al., 2010). In addition, the accumulation of α-synuclein in astroglial cells can cause them to undergo apoptosis (Stefanova et al., 2001). In a mouse model of PD, mesencephalic astrocytes overexpressing α-synuclein show morphological and functional alterations of the glial mitochondria (Schmidt et al., 2011).

Astrocytes are the main site of cerebral ammonia detoxification. The main neuropathological finding in HE is altered astrocyte morphology and the upregulation of GFAP (review, Butterworth, 2009). Astrogliosis may occur during hyperammonaemic conditions (Norenberg et al., 1998; Butterworth, 2003) and after performing PCA (Suárez et al., 1998), and it has been suggested that abnormalities in astrocytes precede any neuronal abnormality in HE (Matsushita et al., 1999). The activation of astrocytes also occurs in the basal ganglia in models of PD, where the increase in GFAP is concomitant with a fall in TH (Rodrigues et al., 2001; Gomide et al., 2005).

The aim of the present work was to determine whether chronic hyperammonaemia affects TH expression and α-synuclein accumulation, as well as GFAP expression in astrocytes in the substantia nigra (SN) and striatum. A progressive reduction in TH expression was seen in both areas, along with an accumulation of α-synuclein in the SNc dopaminergic neurons, and the induction of GFAP expression in astrocytes. Since α-synuclein aggregates also stain for ubiquitin (Ub) in neurodegenerative disorders (Leigh et al., 1991; Mackenzie et al., 2006), the presence of Ub immunoreactivity in SNc neurons was also investigated. The results confirm PCA-induced dopaminergic neurodegeneration in the nigrostriatal system, suggesting α-synuclein accumulationcontributes towards nigrostriatal dysfunction in HE.

2 Material and methods

2.1 Animals and surgery

Two month-old male Sprague Dawley rats were housed under a twelve hour light-dark cycle with free access to food and water in the Lab Animal Resources at the University of Alcalá. Male rats (weighing 180–200 g.) were anaesthetized with halothane and a portocaval anastomosis (PCA) was performed. After surgery, animals were kept on heating pad until recovery from surgery. Shunt patency was confirmed when the animals were sacrificed; all animals had a functioning shunt. Sham-operated rats of the same sex, age and strain served as controls. At designated times (1 month and 6 months after PCA), PCA rats (n = 8 each time point) and sham-operated (n = 8 each time point) were sacrificed for this study. Five rats of each group (1 month and 6 month after PCA and their respective controls) were anaesthetized with halothane and perfused transcardially with saline and subsequently with 3% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and post-fixed in the same solution for 4 hours at 4°C and then the midbrain were isolated, dehydrated through a series of graded ethanol to xylene, infiltrated with paraffin, serially sectioned in 8 μm thick sections and mounted on polylysine coated slides.

One to six months after PCA, three rats of each group and their respective controls were anaesthetized and killed by decapitation, and their left and right substantia nigra and striatum were dissected on an ice-chilled plate. The tissues were stored at –80° C until further procedures were applied. They were used to perform Western blot or dot blot analysis.

All animal experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Alcala Animal Care Committee.

2.2 Immunohistochemistry

Coronal sections (8 μm) were cut through the midbrain of control and PCA exposed rats. Briefly, every 6th section from the region containing the substantia nigra or the striatum was blocked with 5% bovine serum albumin for 30 min at room temperature. The midbrain sections were immunolabelled with antibody to tyrosine hydroxylase (TH) (MAB318, Chemicon, Temecula, CA, diluted 1 : 1000), GFAP (ZO334, Dako, Denmark, diluted 1 : 500), α-synuclein (AB5038, Chemicon,diluted 1 : 1000) or ubiquitin (Ub) (MAB1510, Chemicon, diluted 1 : 500). After three washings, sections were incubated with the appropriate secondary antibodies (1 : 1000; Vector Lab, Burlingame, CA) and visualized using an Avidin-biotin complex (ABC kit, Vector Lab) method and DAB as chromogen (DAB kit, Vector Lab). Sections without primary antibodies were similarly processed to control the unspecific binding of the secondary antibodies. On control sections, no specific immunoreactivity was detected.

Quantification of TH expressing cells: Five animals from each experimental group and control were analysed. Every 6th section of the substantia nigra was immunostained for TH. The number of neurons in SNc was counted using four representative brain sections from each animal and each group. Three microscope fields per section (12 microscope fields per animal) were analysed, all in the same locations and using 40x magnification. Cell count data were analysed by expressing the mean±SEM and comparing the groups (PCA and controls) using the Student’s t-test for unpaired data. The level of significance was set at p < 0.05.

Insoluble a-synuclein immunohistochemistry: Accumulation of insoluble α-synuclein was evaluated by pre-treating the sections with Proteinase-K for 30 min. After quenching endogenous peroxidase with 0.03% H₂O₂ in 70% methanol, the sections were blocked with 5% BSA for 4 h. Thereafter the sections were incubated in α-synuclein (1 : 1000, Santa Cruz Biotechnology Inc, USA) for 72 h at 4°C. This was followed by incubation in biotinylated anti-mouse secondary antibody for 18 h at 4°C (Elite kit, Vector laboratories, Burlingame, USA) and subsequently in avidin–biotin complex. The labelling was visualized using 3% DAB and 0.03% H₂O₂ with nickel sulphate as enhancer. The stained sections were dehydrated in ascending ethanol series, cleared in xylene and mounted in DPX.

2.3 Immunofluorescence staining

Double immunofluorescence staining was performed by simultaneous incubation of sections with two primary antibodies (4°C for 24 h), followed by two secondary antibodies (room temperature for 1 h). Co-localization of TH (1 : 1000) with α-synuclein (1 : 1000), TH (1 : 1000) with GFAP (1 : 500) or α-synuclein (1 : 1000) with ubiquitin (1 : 500) was assessed. Secondary antibodies were FITC-conjugated anti-mouse IgG (1 : 1000, Sigma, CA, USA) and TRICT-conjugated anti-rabbit IgG (1 : 1000, Sigma). After incubation with the secondary antibodies, sections were washed with PBS and mounted using Vectashield mounting medium (Vector Laboratories). Negative controls without primary antibodies were performed for each immunodetection. Other set of sections were rinsed in PBS and then incubated in PBS or Tris containing proteinase K (10 or 20 μg/mL) for 30 min at room temperature. After three washing in PBS, the sections were immunostained with antibodies to α-synuclein (1 : 1000) and ubiquitin (1 : 500).

Fluorescent preparations were examined by using the Leica SP5 laser confocal imaging system. Photomicrographs were captured simultaneously for both fluorophores. Images were imported into Adobe Photoshop 7.0 (Adobe Systems, CA, USA) to compose figures, only adjusted for brightness.

2.4 Western blot and dot blot analysis

Proteins were extracted using CelLytic buffer (Sigma), including 1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor cocktails I and II (Sigma). Lysates were centrifuged at 10,000 g for 12 min and supernatants and pellets maintained at –75°C. Protein concentration was determined using the Bradford microassay (Bio-Rad). Total protein identical amounts of the samples (supernatants) were mixed (n = 3) for every CNS area (SN and ST), time (1 and 6 months) and condition (control and PCA). After electrophoresis (12% polyacrylamide gels), proteins were transferred to Immobilon-P membranes (Millipore). Immunoblot for α-synuclein, GFAP and TH was carried out simultaneously because of the different molecular weight of the proteins. Appropriate anti-IgG peroxidase-conjugated secondary antibodies (1 : 5000) were revealed using DAB (Sigma). Only anti-TH signal could be properly quantified; in fact, anti-α-synuclein signal was very weak and GFAP immunoreactivity appeared as multiple bands. In order to facilitate GFAP determination and considering that α-synuclein could be in an insoluble form, we decide to analyze both proteins using a dot blot analysis.

For dot blot analysis, 6 μg of total protein of the mixed samples were diluted in 100 μL of TRIS buffer and transferred to Immobilon-P using a dot blot apparatus (Bio-Rad). Supernatants were used for GFAP and pellets for α-synuclein. The immunodetection of α-synuclein and GFAP was done in identical conditions to those of the immunoblot previously described. Integrated optical density of the signal on blotting membranes was determined using a videodensitometric procedure (ScionImage).

3 Results

Compared to the control animals, the PCA-treated rats exposed to chronic hyperammonaemia for 1 and 6 months showed modifications in the nigrostriatal system. The neurons of the substantia nigra pars compacta (SNc) showed progressive reduction in TH immunoreactivity plus α-synuclein accumulation. Reduced TH and increased α-synuclein expressions were also seen in the striatum. Increased GFAP expression was also seen in response to PCA in both the SN and striatum.

3.1 Effect of PCA on TH immunoreactivity in the substantia nigra and striatum

In the substantia nigra (SNc), the neurons were strongly TH-immunoreactive in the control animals (Fig. 1a,c,e), but this reactivity became progressively lost in the PCA-treated animals (Fig. 1b,d,f) (11.5±1.1 positive neurons at 1 month and 8.2±2 at 6 months compared to 15.2±2 and 16.9±3 positive neurons for their respective controls; p < 0.05). At 6 months post-PCA (Fig. 1f), the TH immunoreactivity of the SNc neurons was significantly less intense than that seen for the control animals (Fig. 1e). In addition, the typical morphology of the cells was lost following PCA (Fig. 1f). Western blotting showed only a minor decrease in the TH content at 6 months post-PCA (Fig. 4).

Fig.1

Immunohistochemical TH analysis in the Substantia Nigra of controls (a,c,e) and PCA-exposed rats (b,d,f) at 1 month (b) and6 months (d). In PCA exposed brains, the TH immunoreactivity decreased from 1 month (b) to 6 months (d) compared to their respective control groups (a, c). High magnification of TH+ cells in adult controls (e), showing high immunoreactivity in cell bodies and their processes, whereas a reduction in the intensity of TH immunoreactive cell bodies and processes was seen in 6 months PCA exposed brain (f). Representative high-power fluorescent images of striatum showing a reduction in the intensity of TH- positive fibre terminals in 6 months PCA-exposed rats (h) compared with control (g). Scale bars: a-d: 10 μm; e-h: 50 μm.

Fig.2

Substantia Nigra immunostained for α-synuclein in controls (a) and PCA-exposed rats at 1 month (b) and 6 months (c), showing increased α-synuclein expression following PCA exposure. At high magnification, of α-synuclein immunoreactivity was only detected in the neuropil of control rats (d), whereas PCA-exposed rats at 1 month showed some α-synuclein-positive neurons (e), which increased at6 months post-PCA (f). Laser confocal microscopic images of SNc stained with TH and α-synuclein antibodies (g-i). Merged images from adult control (g) and PCA-exposed rats (h-i) No α-synuclein profile was seen in the nigral neurons in controls (g). PCA-exposed rats showed that TH-positive cells were also positive for α-synuclein. Some TH-positive cells showed weak nuclear α-synuclein-immunoreaction at1 month (h, arrows), whereas most of the TH-positive cells at 6 months post-PCA showed high α-synuclein-immunoreactivity in the nucleus (i, arrows). Midbrain sections were double stained for α-synuclein and ubiquitin in controls (j) and PCA-exposed rats at 1 and 6 months (k-l). The merged confocal images demonstrated the presence of α-synuclein and ubiquitin following PCA. Note that the fluorescent intensity of both proteins was increased in the neurons at 6 months post-PCA (l). Coronal section of the dorsal striatum showed weak immunoreactivity in adult control (m), whereas increased α-synuclein immunoreactivity was observed from 1 month (n) to 6 months PCA-exposed rats (o). Scale bars: a-c: 10 μm; d-l: 50 μm; m-o: 200 μm.

Fig.3

GFAP immunoreactivity in the SN of control (a) and PCA-treated rats at 1 month (b) and 6 months (c) showing that PCA treatment induced GFAP activation. Double labelling of GFAP and TH in the SN of controls (d) and PCA-exposed rats (e-f). In comparison with the control SN (d), there was a progressive increase in GFAP with time following PCA (e-f) whereas TH expression decreased in PCA-exposed rats. In the striatum, GFAP and TH double labelling from control (g) and PCA-exposed rats (h-i). Note that GFAP immunofluorescent intensity was increased at 6 months post-PCA (i) concomitant with the reduction in TH expression. Scale bar in a-i: 50 μm.

Fig.4

Western blot (TH) and dot blot (α-syn and GFAP) analysis using a pool of three samples. Samples of substantia nigra (SN) and striatum (ST) were obtained from control (C) and treated (T) rats, at one month (1 m) and six months (6 m) old. Arrow head in the western blot indicates 52 kDa molecular weight marker. Numbers accompanying immunoreactive stripes and dots represent integrated optical density values (arbitrary units).

In the striatum, we analysed the TH immunoreactivity to determine whether the reduced TH expression in the SN dopaminergic neurons of PCA-treated rats was paralleled in their striatal processes. Reductions in striatal TH-positive fibres were observed in PCA-exposed rats, compared to controls (Fig. 1g, 1h), although the loss of TH-immunoreactivity was particularly noticeable at 6 months post-PCA (Fig. 1h). To confirm the reduction in TH expression in the striatal processes, TH was determined in total protein samples extracted from the striatum of PCA-treated (1 and 6 months) and control animals by Western blotting. A reduction in TH was confirmed at 6 months post-PCA(Fig. 4).

3.2 PCA increased α-synuclein immunostaining in the substantia nigra and striatum

The immunoreactivity of α-synuclein in the nigrostriatal system was examined, checking for the presence of aberrant α-synuclein aggregates.Midbrain sections were pre-treated with proteinase-K, since proteinase-K enhances the immunostaining of abnormal α-synuclein.

In the SNc, the neuropil of control animals was immunolabelled with a punctate pattern (Fig. 2a), but no immunoreactivity was seen in the cytoplasm of neuronal or astroglial cells (Fig. 2d). In samples from the PCA-treated animals (Fig. 2b-c), however, an increase was seen over time in the extent and intensity of the punctate pattern, and α-synuclein positive inclusions appeared in the SNc neurons(Fig. 2e-f).

To determine whether α-synuclein accumulated in dopaminergic neurons, the sections were double immunostained with anti-TH and anti-α-synuclein antibodies (Fig. 2g-i). Dual immunofluorescence showed α-synuclein in the SNc dopaminergic neurons as early as 1 month post-PCA (Fig. 2h), but not in the controls (Fig. 2g). A large number of TH-positive dopaminergic neurons in the SNc expressed α-synuclein at 6 months post-PCA (Fig. 2i), although not all TH positive neurons immunostained for α-synuclein. In addition, α-synuclein immunoreactivity was observed in the nuclei of some SNc neurons in the PCA-exposed rats (Fig. 2i). Dot blot analyses confirmed the α-synuclein content to be always higher in the PCA-treated than in the control animals (Fig. 4).

In the striatum, α-synuclein immunostaining was detected in the PCA-treated rats, increasing from 1 to 6 months post-PCA, compared to controls(Fig. 2m-o). This was particularly noticeable in the dorsolateral part of the striatum. Dot blot analysis confirmed the α-synuclein content of the PCA-treated rats to be higher than in the controls, especially at 6 months post-PCA (Fig. 4).

3.3 α-synuclein and ubiquitin immunostaining in the SNc

No Ub staining was detected in the control SNc neurons (Fig. 2j). In the PCA-treated rats, a diffuse accumulation was seen in the SNc neurons at 1 month post-PCA (Fig. 2k), whereas at 6 months post-PCA, some SNc neurons were intensely labelled with Ub (Fig. 2l). The Ub marked cells were also α-synuclein-positive (Fig. 2l).

3.4 Effect of PCA on nigrostriatal astrocytes

In the substantia nigra (Fig. 3a-c), PCA activated the astrocytes, as shown by the increased GFAP immunostaining (Fig. 3b-c) compared to controls (Fig. 3a). GFAP-positive astrocytes appeared to be thicker and more intensely stained at 6 months post-PCA than in controls, with hypertrophic cell bodies and processes forming a dense network (Fig. 3c). The increase in astrocyte GFAP immunoreactivity was concomitant with the reduction in TH expression seen in dopaminergic neurons of the PCA-treated rats (Fig. 3e-f), compared to controls (Fig. 3d). The increase in GFAP expression was confirmed by dot blot analysis: it increased somewhat at 1 month post-PCA, but was much stronger at 6 months post-PCA (Fig. 4).

In the striatum, GFAP-positive astrocytes at 1 to 6 months post-PCA (Fig. 3h-i) showed larger processes than those of control animals (Fig. 3g). The increased expression of GFAP correlated with a reduction in TH staining in the 6 months PCA-treated rats (Fig. 3i).

4 Discussion

In this study, a PCA-exposed rat model of HE was used to evaluate the effect of chronic hyperammonaemia on the nigrostriatal pathway. The results indicate that, in both the SNc and the striatum, PCA causes a reduction in neuronal TH immunoreactivity and an increase in α-synuclein expression. An increase in astrocyte GFAP expression is also seen.

The analysis of TH immunoreactivity in the dopaminergic neurons of the SNc in Parkinson’s disease has revealed these cells to become reduced in number (Mori et al., 2006). In some patients with HE, altered dopamine neurotransmission (Spahr et al., 2002) and signs of PD (Spahr et al., 2000; Layrarges, 2001; Burkhard et al., 2003) have been described, and it has been proposed that the ammonia levels in blood correlate with the severity of HE symptoms (Ong et al., 2003). In PCA-exposed rats, the reduction in TH immunoreactivity in the SN and dorsal striatum progressed with the time of PCA exposure, likely leading to dopaminergic dysfunction since a relationship exists between activity and TH expression in adult mouse SNc neurons (Aumann et al., 2011).

TH expression can be downregulated in adult SNc neurons by exposure to neurotoxins (6-OHDA, MPTP or paraquat) (Vila et al., 2000; Manning-Bog et al., 2002; Gomide, 2005; Matsui et al., 2009). In addition, ammonium chloride treatment reduces TH immunoreactivity in dopaminergic neurons in the medaka fish, causing a movement disorder similar to human PD (Matsui et al., 2010). The loss of TH expression in the dopaminergic neurons of PCA-exposed rats might be related to the high circulating ammonia levels maintained throughout PCA exposure.

Exposure to pesticides caused nigrostriatal degeneration and accumulation of α-synuclein in the substantia nigra in a time-dependent manner (Liu et al., 2015). Moreover, it has been shown that ammonium chloride treatment can induce the formation of α-synuclein-containing inclusions in a human dopaminergic cell line (Matsui et al., 2010). The present results also indicate that chronic hyperammonaemia induced by PCA increases α-synuclein expression in the nigrostriatal pathway, and that this correlates with a reduction in TH expression in dopaminergic neurons and their terminals in the striatum. The question is, how, and to what context is α-synuclein toxic in PCA and/or hyperammonaemia? It has been reported that α-synuclein bind to TH (Perez et al., 2002); it may therefore function as a negative regulator of its expression (Li et al., 2011). It is noteworthy that TH and α-synuclein were seen together in most SN neurons following 6 months of PCA. Elevated α-synuclein might therefore play a role in enhancing vulnerability to dopaminergic dysfunction following hyperammonaemicinsult.

The toxic effects of α-synuclein on dopaminergic cells could be due to the formation of insoluble aggregates of α-synuclein (Yamada et al., 2004). Aggregation of α-synuclein promotes progressive in vivo neurotoxicity in dopaminergic neurons (Taschenberger et al., 2012). Alerte et al. (2008) observed that cells carrying aggregated α-synuclein (as revealed by proteinase-K treatment) showed significantly reduced TH immunoreactivity. Further, proteinase-K-resistant α-synuclein has been described in Lewy bodies, and in presynaptic terminals in PD and DLB (Kramer & Schulz-Schaeffer, 2007; Tanji et al., 2010). In the PCA-treated rats, sections pre-treated with proteinase-K confirmed the presence of insoluble α-synuclein aggregates in the SNc; these were not detected in the control rats. The presence of α-synuclein inclusions in the PCA-exposed rats suggests they showed a neuropathological status, which, together with the loss of TH expression in dopaminergic neurons and impaired motor function, may resemble symptoms similar to early Parkinsonism. When sections were double-stained with antibodies against α-synuclein and TH, the expression of the former was seen to increase while that of the latter decreased in the PCA-exposed rats. Dot blot analysis showed the α-synuclein content to always be higher in PCA-exposed than in the control rats, especially at 6 months post-PCA. Although very little is known about the effects of α-synuclein in HE, ammonia may act synergistically with the protein’s overexpression to induce nigrostriatal cell death ordysfunction.

α-synuclein was also seen in the nuclei of some SNc neurons, the concentration increasing significantly from 1 to 6 months post-PCA. It has been described that the localization of α-synuclein to the nucleus enhances neurotoxicity in dopaminergic neurons (Kontopoulos et al., 2006), and can cause their death (Zhou et al., 2013; Ma et al., 2014). The intranuclear localization of α-synuclein has been reported in MSA (Lin et al., 2004), and in a mouse PD model following intoxication with paraquat (Goers et al., 2003). The same was seen in a dopaminergic cell line following exposure to H₂O₂ (Xu et al., 2006; Zhou et al., 2013). The mechanism by which α-synuclein enters the nucleus, and its function once inside, remain unclear, although it has been shown that α-synuclein can associate with histones (Goers et al., 2003; Kontopoulos et al., 2006) and inhibit their acetylation. In addition, intranuclear co-localization of α-synuclein and Ub was observed at 6 months post-PCA. Intranuclear Ub-positive inclusions are a consistent feature of the major human degenerative diseases (Leigh et al., 1991; Mackenzie et al. 2006), and Ub and α-synuclein are common constituents of Lewy bodies (Gomez-Tortosa et al., 2000; Sakamoto et al., 2002). The presence of both Ub and α-synuclein deposits in the nigral neurons suggests these cells become damaged in PCA-treated brains.

Astrocytes are the main site for cerebral ammonia detoxification; the impairment of ammonia detoxification invariably leads to severe pathologies, and astrocytes undergo morphological changes in the brain of patients with chronic HE (Norenberg, 1998; Butterworth, 2003), as well as in PCA-exposed animals (Suárez et al., 1998). In addition, increased GFAP expression has been reported in the SNc in PD-affected brains (Hirsch et al., 2003; Hirsch & Hunot, 2009), as well as in a rodent model of PD (Ambrosi et al., 2010). In the present work, astrocytes in the SNc and dorsal striatum of the PCA-exposed rats showed hypertrophy in their processes and GFAP upregulation. The increase observed in GFAP immunoreactivity in astrocytes at 1 to 6 months post-PCA was concomitant with the reduction in TH immunostaining in dopaminergic neurons. Gomide et al. (2005) reported that 6-OHDA-induced lesions in the nigrostriatal system induce neuronal and glial alterations: dopaminergic cells showed reduced TH immunoreactivity and reactive astrocytes showed increases in their number and GFAP staining density. Although the normal function of astrocytes was compromised following PCA, as revealed by the increased GFAP expression, PCA treatment induced no increase in the total number of astrocytes in the SN nor striatum, agree with the results reported for the cortex of BDL treated rats (Jover et al., 2006).

It has also been shown in vitro that human astrocyte GFAP reactivity can be caused by α-synuclein (Koob et al., 2010). More recently, α-synuclein aggregation together with GFAP upregulation have been observed in a mouse model of PD (Kurz et al., 2012). In addition, increased numbers of α-synuclein-positive inclusions are reported induced in ammonium chloride-treated brains (Matsui et al., 2010). In the present work, the upregulation of α-synuclein following PCA may have been induced by the circulating ammonia (the major HE-precipitating factor), which might also have activated the astroglial cells and caused the increase in GFAP seen in the dopaminergic pathway. Increases in both GFAP and α-synuclein expression are here shown to be related to a reduction in TH expression. Thus, α-synuclein accumulation might initiate a pathogenic cascade leading to the loss of neurons in PCA-exposed rats, the damage becoming more severe at 6 months post-PCA, with cytoplasmic and nuclear deposits of α-synuclein and Ub appearing in the nigralneurons.

In summary, the present results support the idea that chronic moderate hyperammonaemia induces dopaminergic dysfunction via the downregulation of TH through α-synuclein accumulation in the dopaminergic cells, as well as astroglial activation (as shown by GFAP overexpression). The resulting dysfunctional dopaminergic neurotransmission between the SNc and the dorsal striatum may be the cause of the harmful effects of PCA and/or chronic hyperammonaemia on motor and cognitive function.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Committee.

Footnotes

Acknowledgments

We are grateful to Adrian Burton for reviewing the English version of the manuscript. We thank Mrs. Isabel Trabado for excellent technical support in operating the confocal laser microscope, and the assistance provided by the staff of our animal facility.

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