Original article
Alteration of GSK-3β in the hippocampus and other brain structures after chronic paraquat administration in rats

Introduction

Glycogen synthase kinase-3 (GSK-3) belongs to a family of conserved serine/threonine kinases present in all eukaryotic groups [17]. GSK-3, originally identified as a kinase that phosphorylates glycogen synthase, is rapidly becoming recognized as a central regulator of intracellular signalling cascades because of the wide array of substrates that it phosphorylates [15]. GSK-3 phosphorylates over 50 proteins and is involved in regulation of the cell cycle, cell po­larity, migration, apoptosis and neuroinflammation [21,24,29,63]. GSK-3 has been implicated in the control of cytoskeletal proteins and several transcription factors [52]. The number of cell functions regulated by GSK-3 suggests that the activity of GSK-3 must be tightly regulated. Although the mechanisms regulating GSK-3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK-3-binding proteins [23,29]. GSK-3 consists of two isoforms in humans, namely, GSK-3 (51 kDa) and GSK-3b (47 kDa). GSK-3 and GSK-3b have 97% sequence homology [10]. In this study our attention was concentrated on GSK-3b. It is known that GSK-3b activity is significantly reduced by phosphorylation of an N-terminal serine, Ser9 in GSK-3b and Ser21 in GSK-3 [52]. Several kinases can phosphorylate these serines, including Akt, protein kinase A (PKA), protein kinase C and p90Rsk [11,14]. In op­position to inhibitory regulation by serine phosphorylation, GSK-3 activity is facilitated by phosphorylation of ty­rosine 216 in GSK-3b [20]. This might occur by auto­phosphorylation or by other tyrosine kinases but little is known about regulation of the tyrosine phosphorylation of GSK-3 [4].

The pivotal significance of GSK-3b was justified by previous experimental studies which demonstrated that deletion of the GSK-3b gene in mice is lethal [19,34,59]. The other studies showed that GSK-3b heterozygous (+/–) mice are viable and demonstrated a lot of neurological disorders, for example re­duced exploratory activity, memory consolidation, aggression responsiveness to amphetamine and in­creased anxiety [2,26,41].

Activation of GSK-3b has also been suggested to be involved in a number of degenerative diseases, including Alzheimer’s disease, Parkinson disease (PD) and others, as well as in diabetes type II [23]. Recent studies have pointed to a putative role of GSK-3b in PD [29,58]. Toxins which destroy dopa­minergic neurons selectively, such as 6-OHDA and MPTP, have been found to activate GSK-3b [8,64]. Little is known, however, about the role of GSK-3b in PQ-induced parkinsonism. The controversial non-selective herbicide paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride, PQ), along with other pe­sticides, seems to play an important role in aetio­pathogenesis of PD. Studies on the relationship between PD and pesticides began in the early 1980s, when it was discovered that users of the heroin substitute MPTP, which is chemically similar to paraquat, developed PD. A potential role of PQ in pathogenesis of PD has been suggested because an epidemiological association has been found be­tween its application in agriculture and incidence of this disease [18,32]. The cytotoxic action of PQ clearly involved reactive oxygen species (ROS), an energy crisis, ER stress and neuroinflammation, but its specificity for dopaminergic neurons and possible involvement of the intrinsic cell death pathway are unclear [7,12,16,35,48,55,65-67]. PQ administered in rats acutely induced an increase in extracellular do­pamine metabolism, which suggested an increase in dopamine release [43]. When injected subchronically it reduced the number of dopaminergic neurons in the substantia nigra pars compacta and inhibited dopaminergic transmission [28]. Our last data indicated that long-term PQ administration diversely alters levels of GSK-3b and its active phosphorylated form (pY216) in the midbrain with the pons and striatum, which may be connected with their different vulnerability to PQ toxicity.

Therefore, the aim of this study was to examine the influence of long-term (37 weeks) PQ administration in rats on the levels of total GSK-3b and its active form, phosphorylated on tyrosine 216 (pY216) in the hippocampus, brain cortex and cerebellum. Since GSK-3b has been shown to be located in several cell compartments such as cytosol, nucleus and mitochondria (using microscopy and immunoblotting experiments) [5,42,46,49,58], the subcellular loca­-lization of this kinase was analysed.

Material and methods

Animals



The experiments were carried out in compliance with the Animal Protection Bill of August 21, 1997 (published in Journal of Laws no. 111/1997 item 724), and according to the NIH Guide for the Care and Use of Laboratory Animals. They also received the approval of the Local Ethical Committee. All efforts were made to minimize the number and su­ffering of animals used. Male Wistar rats 3 months old weighing 200-250 γ at the beginning of experiments were kept on a light/dark cycle (12/12 h; the light on from 7 am to 7 pm) with free access to food and water.

Drug

Paraquat dichloride (Sigma-Aldrich, Germany) was dissolved in sterile water and administered at a dose of 10 mg/kg/2 ml i.p. once a week for 37 weeks. The dose of paraquat was chosen according to earlier papers [28,43-45,58]. Animals were killed by decapi­tation 7 days after the last injection. Control animals were treated with physiological saline i.p. once a week.

This model was described previously by Songin et al., 2011 [58].

Tissue sectioning

After decapitation, brains were rapidly removed and sectioned on an ice-cold Petri dish. Hippocampus, brain cortex and cerebellum were analysed by Western blotting (GSK-3b).

Isolation of subcellular fraction

After dissection, the hippocampus, brain cortex and cerebellum were immediately frozen and stored at –80°C until further procedures were applied. The structures were homogenized in 0.32 M sucrose with 10 mM Tris-HCl, pH 7.4, 1 mM EDTA (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and with Complete™ protease inhibitors cocktail (Roche Ap­plied Science, Indianapolis, IN, USA)]. The 10% ho­mo­­ge­nates were centrifuged at 900 γ for 3 min at 4°C. The pellet (P1)-crude nuclear fraction was resuspend­ed in the above described solution. The supernatant (S1) was centrifuged at 15,000 γ for 10 min at 4°C to separate the crude mitochondrial fraction (P2) and cytosolic fraction (S2). The pellets (P1, P2) were resus­pended in 10 mM Tris-HCl (pH 7.4) buffer with protease inhibitors. Protein concentration was determin­ed by protein dye-binding with a commercial reagent (Bio-Rad Laboratories, Hercules, CA, USA). Then crude nuclear, mitochondrial and cytosolic fractions were obtained from each brain part and mixed with 5 × Laemmli sample buffer and denatured for 5 min at 95°C. After standard SDS-PAGE on 10% poly­acryla­mide gel, proteins were transferred onto PVDF membranes, then the membranes were washed for 5 min in TBS-T buffer (20 mM Tris, 500 mM NaCl, pH 7.5, TBS containing 0.5 ml/L Tween 20). After incubations with antibodies and autoradiography, densitometric analysis and marker size-based verification were performed with TotalLab software.

The immunochemical determination of a typical mitochondrial protein, apoptosis inducing factor (AIF), indicated that this protein was localized exclusively in the mitochondrial fraction of control and PQ-treated animals. Our previous study [60] indicated that DNA-bound enzyme (ADP-ribose) polymerase-1 (PARP-1) was localized exclusively in the nuclear fraction and has never been detected by us in mitochondria or in cytosol using a similar procedure for the isolation of subcellular fractions. In the aged striatum, the level of PARP-1 was significantly lower, which may suggest specific sensitivity of this brain part to age-related processes [60].

Electrophoresis and immunoblotting

The electrotransfer of proteins to PVDF membra­­nes was performed for 1.5 hours at 100 V and then the membrane was blocked for 1 h at room temperature in 5% non-fat milk in TBS-T for GSK-3b or in 0.5% BSA/TBS-T for GSK-3b (pY216). After washing in TBS-T, the membranes were immuno­-stained according to standard methods provided by the manufacturer (BD Transduction Laboratories, San Diego, CA, USA). They were probed with a primary mouse monoclonal anti-GSK-3b antibody (1 : 2,500) in 5% non-fat milk/TBS-T or anti (pY216)-GSK-3b IgG (1 : 1000) in 0.1% BSA/TBS-T (BD Transduction Laboratories, San Diego, CA, USA), were incubated overnight (4°C), washed three times in TBS-T and were further incubated with the secondary anti-mouse antibody conjugated with horseradish peroxidase (HRP) (1 : 4,000) (Amersham) in 2% non-fat milk/TBS-T. After stripping, membranes were probed with mouse monoclonal anti-beta-actin antibody (1 : 400) in 0.1% BSA/TBS-T to normalize protein levels (MP Biomedicals, Inc, Irvine, CA, USA). The blots were developed using the en­hanced chemiluminescence (ECL) detection system (Amersham International, Aylesbury, U.K.) according to the manufacturer’s protocol. After exposure, photographs were taken with a Kodak DC 290 zoom digital camera and were analysed using the Kodak EDAS 290/Kodak 1D 3.5 system.

Statistical analysis

We used Student’s t-test for Western blotting; p  0.05 was considered significant. The presented data are means ± SEM.

Results

The influence of the long-term paraquat admini­stration on the levels of GSK-3b and GSK-3b (pY216) in nuclear, mitochondrial and cytosolic fractions in the hippocampus, brain cortex and cerebellum.

Our data confirmed the presence of both total and active (pY216) form of GSK-3b in nuclear, mitochondrial and cytosolic fractions. The results present­ed in Fig. 1 demonstrate that paraquat administered in a dose of 10 mg/kg i.p. for 37 weeks influenced levels of that enzyme in the brain structures exa­mined.

Hippocampus

PQ significantly decreased the total level of GSK-3b immunoreactivity in isolated nuclear (P1) and cytosolic fractions (S2) of the hippocampus. The level of GSK-3b (pY216) was also significantly lowered in the same fractions of this region (Fig. 1A).

Brain cortex

In the brain cortex, both total and phosphorylated (pY216) GSK-3b immunoreactivity levels were decreased in the nuclear fraction in rats treated with PQ (Fig. 1B). In contrast, the level of both forms of GSK-3 was increased significantly in the mitochondrial fraction and that of GSK-3b (pY216) in the cyto­solic fraction. Moreover, a similar trend (but insig­nificant; p = 0.09) in the cytosolic fraction was observed for the total form of GSK-3 (Fig. 1B).

Cerebellum

In the cerebellum, the immunoreactivity levels of both total and active form of GSK-3 were significantly decreased in the nuclear fraction, whereas they were increased in the mitochondrial fraction after PQ (Fig. 1C). PQ additionally increased the level of total GSK-3 in this structure in the cytosolic fraction, but decreased slightly but significantly the phos­phorylated form of this enzyme (Fig. 1C).

Discussion

This study showed that the long-term administration of PQ in rats diversely influenced the levels of GSK-3b in different brain structures. We found that PQ lowered levels of both total and active (pY216) forms of this kinase in the nuclear and cytosolic fractions in the hippocampus. In contrast, in the brain cortex and cerebellum this pesticide decreased levels of GSK-3b and GSK-3b (pY216) in the nuclear fraction, but increased them in the mitochondrial fractions and in some cases also in the cytosol, which suggested translocation of this kinase from the nucleus to other cellular compartments.

In our previous paper [58], we demonstrated that the same 37-week regime of PQ administration as used in the present study increased the level of GSK-3b and its active (pY216) form in all cellular compartments in the midbrain and pons, and induced an opposite effect in the striatum. All these results showed that the long-term exposure to PQ not only influenced variably the level of GSK-3b but also affected its activation in different brain structures. These alterations may have significant implications for cell functions.

Our previous papers have shown that long-term 24-37-week treatment with PQ moderately de­creased the number of dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area, as well as noradrenergic cells in the locus coeruleus, and weakly lowered the level of dopamine, or its metabolism in the striatum in rats [43-45,58]. These results were in good agreement with several others which showed that PQ induced loss of dopaminergic neurons and disturbances of dopaminergic transmission [6,25,28,37,57]. Our last paper also suggested that increased levels of the total and activated (pY216) form of GSK-3b in the midbrain and pons may be involved in the PQ-induced degeneration processes progressing in cate­cholaminergic neurons [58]. Such a role of GSK-3b may be supposed because this kinase contributes to the production of reactive oxygen species [62] and its overexpression promotes cell death caused by the p53-dependent mitochondrial intrinsic apoptotic pathway [3,31], which are the main mechanisms responsible for the PQ-induced toxicity [66]. Moreover, recent studies suggested that microglia-dependent inflammatory processes (including activation of pro-inflammatory cytokines and nuclear factor [NF]-B) contribute to PQ-induced toxicity towards dopaminergic neurons [35,48]. GSK-3b is known as a regulator of inflammation via its influence on NF-B [24,63] and therefore the activation of this kinase in the midbrain and pons [58] is likely to be involved in neuroinflammation induced by PQ in these regions. Further support for a crucial role of GSK-3b for de­generative processes in catecholaminergic neurons comes from studies of other authors who showed that activation of this kinase (via increased phosphorylation on tyrosine 216 or decreased phosphorylation of serine 9) contributed to the promotion of cell death caused by other PD-related toxic agents, such as 6-hydroxydopamine (6-OHDA), rotenone and MPTP [8,27,64] and was involved in proteasomal inhibition [1].

Selective toxic influence of PQ on dopaminergic neurons in mice after its systemic administration was postulated by the earliest studies [37]. How­ever, levels of this pesticide are elevated in parallel with increasing number of injections [47]. This may lead to its deleterious action on non-catecholaminergic neurons after prolonged (37-week) administration, as well. In agreement with this view, repeated injections of PQ alone or in combination with the fungicide maneb in normal and/or -synuclein mutant mice were found to induce histopathological alterations (e.g. changes in neuronal arrangement, presence of pyknotic nuclei, disorganization of mitochondrial membrane and others) in neurons of se­­veral brain structures including hippocampus and cerebellum [9,40]. Our present study showing an influence of PQ on levels of GSK-3b in the hippo­campus, cerebral cortex and cerebellum also indicates a rather non-selective, widely spread action of PQ throughout the brain. However, the mechanisms underlying these alterations are not understood and at this stage of study it is difficult to judge what kind of consequences they could cause.

PQ is known to destroy the cytoskeleton of different cells, including neurons, and has been found to induce microtubule aggregation and bundling, microfilament redistribution, as well as axonopathy, measured by a decrease in the number of neurofilaments [30,39,53,54,56]. All these alterations may disturb axonal transport. Our previous study [45,58] showed that PQ destroyed the sources of catecholaminergic hippocampal innervations: the no­radrenergic locus coeruleus [36] and the dopaminergic ventral tegmental area [61]. Therefore, it seems possible that PQ also destroyed transportation of GSK-3b and GSK-3b (pY216) from catecholaminergic cell bodies in the above regions to their terminals in the hippocampus, which resulted in the lower levels of this kinase in nuclear and cytosolic fractions of the latter structure. Similar processes related to dege­neration of the dopaminergic nigrostriatal pathway could be responsible for the decrease in levels of GSK-3b and its active form in the striatum reported in our previous paper [58]. However, in the light of the aforementioned signs of neuropathology in the hippocampus induced by PQ administration [9,40], it is highly probable that alterations of GSK-3b in this structure could be an adaptive, protective reaction activated concomitantly with degenerative processes triggered by this pesticide. The appearance of such processes in response to several neuronal toxins is a well-known phenomenon and with regard to PQ recent studies have shown activation of both pro- and antiapoptotic processes in cell lines after low doses of this pesticide [51]. Since some earlier studies have demonstrated changes in neurotransmissions in the hippocampus induced by PQ which were dependent on IFN-, it is likely that reduction of GSK-3 levels and its activation could oppose the development of inflammation in this structure [33].

However, in other brain structures, such as the brain cortex and cerebellum, 37-week PQ administration changed the subcellular localization of GSK-3 and GSK-3 (pY216), which were translocated from the nucleus to the mitochondria. Since GSK-3 is a multifunctional kinase [3], the consequences of these alterations are unclear at present. Although GSK-3 is located predominantly in the cytosol, activities of this enzyme in the nucleus and mitochondria are much greater than in the cytosolic fraction [5] and every redistribution of this enzyme between the subcellular organelles may be of special importance for its function. The translocation of GSK-3 from the cytosol to the nucleus or mitochondria is known to appear during apoptotic conditions [3]. GSK-3 influences a large number of transcription factors that control gene expression of apoptotic and anti-apoptotic proteins, as well as proteins involved in cellular growth and survival, neuroinflammation, or oxidative stress [3,13,22]. Therefore the reverse process, a decrease in the level of GSK-3 in the nucleus (which was observed in all examined brain structures except the midbrain and pons after PQ), may diminish its transcription activity and in this way may affect both degenerative and protective compensatory processes developing in response to this herbicide [22]. On the other hand, PQ is a toxin which disrupts mitochondrial structure and several of its functions [12,38,40,50,66]. Therefore, increased levels of GSK-3b and GSK-3b (pY216) in this subcellular compartment in brain cortex and cerebellum may facilitate these processes [3]. Finally, it seems that the fate of neurons in the above-mentioned brain structures after PQ administration may depend on the result of alterations of all processes which involve GSK-3b.

Summing up, the present paper suggests that long-term PQ administration influences levels and activation of GSK-3 in different brain structures, which may contribute to its toxicity, but on the other hand may suggest development of adaptive, protective processes.

Supported by Scientific Network of MNiSW No. 28/E-32/BWSN-0053/2008.

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