Involvement of heat shock proteins on Mn-induced toxicity in Caenorhabditis elegans

All living cells display a rapid molecular response to adverse environmental conditions, and the heat shock protein family reflects one such example. Hence, failing to activate heat shock proteins can impair the cellular response. In the present study, we evaluated whether the loss of different isoforms of heat shock protein (hsp) genes in Caenorhabditis elegans would affect their vulnerability to Manganese (Mn) toxicity. We exposed wild type and selected hsp mutant worms to Mn (30 min) and next evaluated further the most susceptible strains. We analyzed survival, protein carbonylation (as a marker of oxidative stress) and Parkinson’s disease related gene expression immediately after Mn exposure. Lastly, we observed dopaminergic neurons in wild type worms and in hsp-70 mutants following Mn treatment. Analysis of the data was performed by one-way or two way ANOVA, depending on the case, followed by post-hoc Bonferroni test if the overall p value was less than 0.05. We verified that the loss of hsp-70, hsp-3 and chn-1 increased the vulnerability to Mn, as exposed mutant worms showed lower survival rate and increased protein oxidation. The importance of hsp-70 against Mn toxicity was then corroborated in dopaminergic neurons, where Mn neurotoxicity was aggravated. The lack of hsp-70 also blocked the transcriptional upregulation of pink1, a gene that has been linked to Parkinson’s disease. Taken together, our data suggest that Mn exposure modulates heat shock protein expression, particularly HSP-70, in C. elegans. Furthermore, loss of hsp-70 increases protein oxidation and dopaminergic neuronal degeneration following manganese exposure, which is associated with the inhibition of pink1 increased expression, thus potentially exacerbating the vulnerability to this metal.


Background
Molecular chaperones are highly evolutionarily conserved and ubiquitously found in subcellular compartments, cells, and tissues, being essential for the stability of the proteome under normal and stressful conditions [1]. The expression of many molecular chaperones is regulated by environmental and physiological stresses that can interfere with folding stability, leading to a flux of misfolded proteins [2]. Stress responsive molecular chaperones are referred to as heat shock proteins (HSPs) and classified by gene families according to their molecular mass as Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and small Hsps (sHsps). HSPs exert their physiological effect by assisting the formation of new proteins as well as by preserving existing structures. However, they also display major functions in pathological conditions, especially through structural rectification of denatured proteins and solubilization of protein aggregates carrying them on to the proteasome system [2,3].
Metal exposure at different levels can cause oxidative stress, which can lead to protein aggregation [4,5]. Thereby, metals themselves are able to generate aberrant interactions with proteins such as beta-amyloid, α synuclein and prion proteins [5,6]. In this context, Manganese (Mn) poisoning has been associated with increased heat shock protein levels, especially HSP70 [7][8][9]. Mn is widely used in industry and in agriculture, being found in several products such as batteries, pesticides, gasoline, parenteral nutrition, water purification agents and drugs [10][11][12][13][14]. As a consequence, exposed subjects may develop a syndrome known as manganism, where alterations in movement, speech and face expression may appear [14][15][16].
Manganism and Parkinson´s disease (PD) share several symptoms and molecular mechanisms [17]. Several lines of evidence point out that the behavioral and cognitive impairments are due to the dopaminergic alterations in brain areas that are involved in the movement circuitry. Depletion of dopamine (DA) from the dopaminergic (DAergic) neurons, mitochondrial dysfunction, oxidative stress and neuronal death have been reported in both disorders. While PD is mostly idiopathic in its etiology, many genes have now been associated with the disease called as PD-related genes. The mutation of dj-1, pink-1, parkin, for example, have been strongly linked to the early-onset of PD neurodegenerative disease in humans. In addition, it has been demonstrated that mutation in these genes also relate to onset of manganism [18][19][20]. As a putative treatment, recently, studies provided evidence on the important role of HSP70 in recovering DAergic neurons or degrading misfolded proteins in PD models [21,22].
In order to study the involvement of HSPs on Mninduced neurotoxicity, we used the Caenorbabditis elegans model. This nematode incorporates 302 neurons, 8 of them being dopaminergic. Its transparent body and ease of genetic manipulability turns it into very interesting model to study neurodegeneration and to unravel molecular targets of toxicants. Our group has already demonstrated that Mn causes specific degeneration in the dopaminergic neurons through molecular mechanisms that replicate the effects observed in mammalian models. Furthermore, C. elegans possess 21 isoforms of HSP, which have been shown to function as chaperones and to have antioxidant role in worms as well as in mammals [23,24].
Hence, we hypothesized that the deletion of hsp genes would increase Mn-induced oxidative stress and DAergic neurotoxicity. Furthermore, we investigated whether this deletion would affect the expression of some PDrelated genes, based on the hypothesis that HSPs might be carriers of DJ1, PDR1 and PINK1 to the mitochondria following Mn stress.

Chemicals
Oxyblot protein oxidation analyses kits were purchased from Millipore (S7150-Billerica, CA). All the other reagents were obtained from Sigma (St Louis, MO).

Dose-response curves after Mn exposure
Five thousand synchronized L1 stage worms per dose were treated for 30 min with each of the compounds, followed by three washes with 85 mM NaCl soultion. Worms were placed on OP50 seeded NGM plates and the dose-response curves were plotted from scoring the number of surviving worms on each dish at 24 h postexposure. Dose response curves and LD 50 values were obtained from those curves. Worms were then exposed for 30 min to 35 mM manganese chloride (MnCl 2 ), which corresponds to the LD 25 for MnCl 2 as previously reported by Benedetto et al. [27]. For all dose-response curves, scores were normalized to percent control (0 mM MnCl 2 exposure).

Confocal microscopy
For each slide, at least 20 worms were mounted on 4 % agarose pads in M9 and anaesthetized with 0.2 % tricaine/ 0.02 % tetramisole in M9. Fluorescence observations were performed with an epifluorescence microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) equipped with a Lambda LS Xenon lamp (Sutter Instrument Company) and Nikon Plan Fluor 20× dry and Nikon Plan Apo 60 × 1.3 oil objectives. Microscopes were housed in air-conditioned rooms (20-22°C). Worms were observed 2 h after Mn exposure.

Real time PCR
Total RNA was isolated using the TRIzol reagent (Invitrogen) and the RNeasy mini kit (Qiagen). First-strand cDNA synthesis was performed with an equal amount of RNA using the Thermoscript real-time PCR kit (Invitrogen) as per the kit's instructions. The genes observed through real-time PCR were as follows: hsp-70, pdr-1, dj-1, parkin ( Table 1). The housekeeping gene act-1 was used as an internal control. Primer sequences are available on request. mRNA expression was quantified using the SYBR green detection method on an Bio-Rad real-time PCR system. Relative quantification for the expressed genes was done using the comparative C T (ΔΔC T ) method.

Statistics
Dose-response lethality curves, longevity curves and ROS content and oxyblot analysis were generated with GraphPad Prism (GraphPad Software Inc.). We used a sigmoidal dose-response model with a top constraint at 100 % to draw the curves and determine the LD 50 or the average lifespan values reported in the graphs. Statistical analysis of significance was carried out by one-way or two way ANOVA, depending on the case, followed by post-hoc Bonferroni test if the overall p value was less than 0.05. In all figures, error bars represent the standard errors of the mean (SEM).

Results
The loss-of-function of some HSP genes led to increased sensitivity towards Mn (Table 2). While Mn-induced lethality of the transgenic strains lacking hsp-4, hsp-43, hsf-1 and hsp-12.6 were indistinguishable from wild type worms, hsp-70 mutants exhibited hypersensitivity to Mninduced lethality (LD50 = 73.08 mM) compared to N2 worms (LD50 = 46.13 mM) (Fig. 1a, p < 0.05). Conversely, other genetic deletions caused decreased Mn-induced lethality in comparison to wild type worms such as hsp-3 (which has 99.1 % homology to HSPA5 from the HSP70 family, Fig. 1b) and chn-1 (homologous to CHIP (Cterminus of Hsc70 interacting protein), Fig. 1c). In order to identify the possible underlying mechanisms, we determined the extent of Mn-induced oxidative damage through an indirect method: the measurement of protein carbonyl content. We observed that all strains showed increased carbonylation at lower Mn concentrations in comparison to N2, corroborating the findings of the survival assay (Fig. 2a,  b and d). Notably, the lack of HSF-1 neither change Mn toxicity (Fig. 1d), nor increased carbonyl content (Fig. 2c).
Considering the higher sensitivity of hsp-70 worms following Mn exposure, we generated transgenic worms lacking hsp-70 and expressing pdat-1::GFP. The expression of the green fluorescent protein (GFP) under the control of a promoter for the dopamine (DA) re-uptake transporter 1 allows the visualization of the architecture of the DAergic neurons. Following Mn exposure the pdat-1::GFP fluorescence and morphology of the mutants lacking hsp-70 was compared to wild type (N2) (Fig. 3). The neurodegeneration induced by Mn in wild type worms occurred as previously reported by Benedetto et al. [27]. Remarkably, the knockout of the hsp-70 gene caused significant degeneration in DAergic neurons following 10 mM (ballooning of the neurons soma) Mn exposure (Fig. 3). Corroborating the importance of hsp-70 gene as a Mn-responsive gene, we further observed that the mRNA expression of this gene increases significantly with increased Mn concentrations (Fig. 4).
In order to evaluate the effect of Mn exposure on the expression of PD-related genes in wild type and hsp-70 mutants, we determined the mRNA levels of pdr-1, djr-1.1 and pink-1. Increased Mn concentrations applied to WT worms led to a dose-dependent increase in the expression of pdr-1, djr-1.1 and pink-1 (Fig. 5). Unexpectedly, mRNA levels of pdr-1 and djr-1.1 remained increased in hsp-70 mutants following increased Mn concentrations (Fig. 5a and c). However, two-way ANOVA revealed a strong interaction between Mn concentration and genotype, supporting the notion that the dynamics of the transcriptional response to Mn exposure differs between wild type and hsp-70 worms. In particular, hsp-70 mutation abrogates the dose-dependent increase in pink-1 expression typically observed upon graded Mn exposure (Fig. 5b). Interestingly, this suggest that HSP-70 is specifically required for the Mn-induced increase in PINK-1 expression.

Discussion
The cellular and molecular evolutions of most organisms rely on HSPs, which promote cell adaptation and survival under conditions of stress [2]. In the present study, we used an environmental and occupational metal toxicant and observed that the absence of hsp-70 gene leads to increased sensitivity to Mn-induced neurotoxicity, which was associated with increased oxidative stress and specific alterations in mRNA levels of pink-1 in C. elegans. Other hsp genes may also be related to Mn-induced neurotoxicity. However, worms lacking hsp-4, hsp-43, hsf-1 and hsp-12.6 were less sensitive towards Mn-induced toxicity as compared to nematodes lacking hsp-70. hsp-70 encodes a member of the HSP70 family genes in C. elegans with 13-members identified in the organism so far [29]. It comprises approximately 100 % homology to the human HSPA8, an isoform of heat shock cognate of 71 kDa (hsc70) [24]. The hsp-70 gene is under normal conditions expressed constitutively throughout nematodes development. With increasing temperatures hsp-70 mRNA synthesis is enhanced 2-6-fold [24,30]. In mammals a large variety of cellular functions have been attributed to HSPA8 most of them through its cooperation with co-chaperones. Thereby HSPA8 participates in the chaperone-mediated autophagy, an important process that recognizes malformed proteins in order to degrade them through the ubiquitin/proteassome system [31].
Mn exposure modulates the expression of HSPs in different species [7][8][9][32][33][34][35]. Most of the evidence points out to an increase of HSP-70 levels following acute Mn exposure, thus reflecting an attempt to protect from the toxic and pro-oxidative effects triggered by Mn [7][8][9]32]. This is corroborated herein, as the absence of hsp-70 led to higher vulnerability to Mn exposure, which was   [37]. In addition, in vitro and in vivo studies demonstrated that Hsp70 might play a role in neuroprotection against MPTP and rotenone (two models of PD) by inhibiting pro-apoptotic factors as well as by activating survival pathways [38][39][40]. PDrelated genes DJ1, parkin and Pink1 are participating in the oxidative stress response and protect the cell against mitochondrial oxidative stressors such as Mn.
Regarding the gene DJ1, the nematode expresses two orthologues named djr-1.1 and djr-1.2. Recently, our group demonstrated that pdr-1 and djr-1.1 loss in C. elegans increased their susceptibility to Mn in comparison to wild type worms and that the observed enhanced oxidative stress is related to increased Mn accumulation [19]. In addition, the higher Mn accumulation caused by loss of pdr-1/parkin gene was due to reduction of ferroportin (a Mn cell exporter) expression in worms [20]. Furthermore, Chen et al. demonstrated that worms overexpressing DJR-1.2 are not subject to lifespan reduction caused by Mn exposure, in contrary to djr-1.2 mutants [41]. Interestingly, under stress conditions, DJ-1 is translocated to mitochondria by HSP-70 [42]. It was further shown that a bcl-2 associated Athanogene 5 (BAG5) can enhance DAergic neuronal death by inhibiting both Parkin and the chaperone activity of Hsp70 [43]. Hence, we hypothesized that the absence of hsp-70 would alter the expression of these genes. First, we verified for the first time that Mn increases mRNA levels of all these genes in wild type worms, which is in agreement with the hypothesis that these proteins are required to protect cells against Mninduced toxicity [19,20,41]. While in the hsp-70 mutants mRNA levels of pdr-1 and djr-1.1 increased dosedependently in a manner indistinguishable from N2 worms, the pink1 expression failed to increase and was not significantly different compared to non-treated mutants. Pink1 (PTEN-induced kinase 1) is a mitochondrial kinase consisting of 581 aminoacids that encode a mitochondrial targeting sequence, a transmembrane domain and a Ser/Thr kinase domain. PINK1 is believed to confer neuroprotection by policing mitochondrial integrity [44] and a growing amount of data links dysfunction of mitochondrial dynamics with PD  [45,46]. Hence, loss of pink-1 is associated with mitochondrial impairments, oxidative stress, and DAergic neuronal loss, as DA neurons may be particularly vulnerable to mitochondrial dysfunction [47,48]. A proteomic study of Triplett et al. with PINK1 knockout mice showed that these animals have reduced HSP-70 levels in their brain [49]. Herein, we observed that hsp-70 mutants blocked pink-1 -mRNA expression following Mn exposure compared to the respective dosedependent increase observed in WT worms, which reinforces the relationship between these two genes. In accordance, as we observed a significant impairment of DAergic neurons in the worms lacking hsp-70, we can infer that the mitochondrial dysfunction provided by Mn, plus absence of an important chaperone and the failure of the cells to increase PINK1 expression would culminate with the higher damage to these neurons. Constructing a worm that overexpress PINK1 in a hsp-70 KO background would give us a more reliable view on the role of pink-1 and hsp-70 in Mn-induced DAergic degeneration. Working with gene profiling in C. elegans, which can be visualized in vivo using transgenic GFP-tagged strains, Anbalagan et al. demonstrated that Cd 2+ , Cu 2+ , Hg 2+ and Zn 2+ exposure induce the heat shock genes quite strongly (hsp-16.1, hsp-16.2, hsp-6, hsp-60 were at least 2-fold increased) [50]. Notably, we observed that Mn exposure increased mRNA levels of hsp-70 (Fig. 4) and that HSP-4::GFP and HSP-6::GFP levels were significantly increased following Mn exposure, reinforcing the fact that this metal can indeed modulate these chaperones (data not shown).
We also observed that the absence of other chaperones as hsp-3 and chn-1 led to increased Mn-induced toxicity including compared to wild type woms. hsp-3, is expressed constitutively and is non-heat inducible; its mRNA is most abundant at the L1 larval stage [24]. Since mRNA of hsp-3 is found at maximum levels in the L1 stage [30], it might be very important to protect the larvae against toxicants. chn-1 is the homologue of the human CHIP, which is very important for removing defective and misfolded proteins. Springer et al. demonstrated that CHN-1 forms a protein complex with PDR1/Parkin, in order to ubiquitylate proteins [51]. Hence, loss of chn-1 already causes issues in worms development [52]. Consequently, we decided not to pursue further investigations into DAergic neuronal vulnerability in these two mutants. Interestingly, loss of hsf-1 (heat shock factor) did not cause significant vulnerability to Mn exposure (Fig. 1d). This is not surprising because it has been demonstrated that HSF-1 is not the only transcription factor that activates HSPs expression. In fact, it has been demonstrated that DAF-16 and SKN-1 can also modulate the transcription of some HSPs [53,54].

Conclusions
Taken together, our data suggest that Mn exposure modulates HSP expression, particularly HSP-70, in C. elegans. Furthermore, loss of hsp-70 prones worms to increased  protein oxidation and increased DAergic neurodegeneration following Mn exposure. This might be associated with a blockage of the pink1 expression, which can hypothetically exacerbate mitochondrial dysfunction caused by Mn exposure since pink-1 expression is normally increased in wild type worms following Mn exposure. Consequently the current study provides evidence for the neuroprotective role of hsp-70 in Mn-induced neurotoxicity and a possible protective role of overexpressing hsp-70 needs to be clarified in future studies.