Adult (eight to ten weeks) male C57/BL6 mice (Janvier Breeding Centre, France) were housed, handled, and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (NCR (National Research Council) 1996) and the European Union Council Directive 86/609/EEC, and the experimental protocols were carried out in compliance with institutional ethical committee guidelines for animal research. For all studies, mice were maintained on a 12:12 hour light/dark cycle with lights on at 6.30 am. The room temperature was kept at 23°C, with free access to standard diet (LASQCdiet® Rod16-R, LASvendi) and tap water. Mice (n = 8 to 10 per group) were treated using the acute MPTP paradigm (4 × 20 mg/kg MPTP-HCl (Sigma-Aldrich, Lyon, France) every 2 hours) 20 21 and animals were sacrificed after 12, 24, 48, 96 or 168 hours after the last injection. The brain was excised and the specific brain regions dissected. The corresponding controls were collected variously across the days.

Striatum and SN of the mice were dissected. Tissues were then placed into Trizol reagent (Invitrogen, Cergy Pontoise Cedex, France), homogenized, and total RNA was prepared according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using the ThermoScript RT-PCR system (Invitrogen, Cergy Pontoise Cedex, France). To access expression levels specific designed intron-spanning primers for Hsp60 (fwd = 5′-CAC AGT GAA GGA TGG AAA AAC CCT-3′ and rev = 5′-TCT TTG GTG ACA ATG ACC TCC C-3′) and tubulin (fwd = 5′-TGT CCA TGA AGG AGG TGG ATG AG-3′ and rev = 5′-ATG TTG CTC TCA GCC TCG GTG AAC-3′) were used. The annealing temperature was 64°C and the number of PCR cycles was chosen to stop the reaction in the linear phase of amplification (25 cycles). The amplification of Hsp60 and tubulin was done in the same reaction for each sample. After gel electrophoresis, digital images were analyzed with NIH ImageJ software for quantification.

The Hsp60 and TH primers for quantitative PCR were designed by primer express 3.0 (Applied Biosystems, Darmstadt, Germany) and their specificity was confirmed by blastn analysis. Their sequences were as followed: Hsp60_fwd: 5′-GCC GCC CCG CAG AA-3′, Hsp60_rev: 5′-CCT GGA CAC CGG TCT CAT CT-3′, TH_fwd: 5′-GCA CCT TCG CGC AGT TCT-3′, TH_rev: 5′-ACA GCG TGG ACA GCT TCT CA-3′. Mesencephalon ss PD patients and age-matched control subjects (n = 5 per group) were provided by the INSERM UMR_S1127 brain bank. The SN was dissected from frozen slides. Total RNA was extracted with Trizol (Invitrogen, Cergy Pontoise Cedex, France) and the quantity and quality were determined by Nanodrop and Agilent, respectively. One microgram of total RNA was reverse-transcribed with Superscript III RT-kit (Invitrogen, Cergy Pontoise Cedex, France) according to the manufacturer’s instructions. qPCR was performed with SYBR GreenER TM qPCR SuperMix kit (Invitrogen, Cergy Pontoise Cedex, France) in ABI 7500 real time thermal cycler (Applied Biosystems, Darmstadt, Germany). The relative mRNA expression levels were normalized by the geometric mean of two housekeeping genes (GAPDH and HPRT) with qBaseplus software (Primerdesign, Southampton, UK).

SH-SY5Y (p3-p7) cells 22 were maintained in DMEM (Gibco, Saint Aubin, France) supplemented with 15% FCS, 1 mM L-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin sulfate/ml and equilibrated with 5% CO₂-95% air at 37°C. At different time points, the cells were treated with 100 μM MPP⁺ (1-methyl-4-phenylpyridinium) 23 purchased from Sigma-Aldrich (Lyon, France).

Mesencephalic cultures were prepared from the ventral midbrain of Swiss mouse embryos at gestational age 13.5 days (Janvier Breeding Center, France). The dissected tissue pieces were processed according to previously described protocols 24 25 . Briefly, after mechanical dissociation in modified L15 medium 26 with no enzymatic treatment, the cells in suspension were plated at a density of 1.5 to 2.0 × 10⁵ cells/cm² in polyethylenimine (PEI, Sigma-Aldrich, Lyon, France; 1 mg/mL) precoated culture plates (24 well-plates). The cells were then allowed to mature and differentiate in N5 culture medium 25 27 , supplemented with 5% horse serum and 0.5% fetal calf serum (FCS, endotoxin level of the used FCS < 10 EU/ml) except for the first three days in vitro (DIV), when the concentration of FCS was raised to 2.5% to favor cell attachment and initial development. Mesencephalic DA neurons degenerate spontaneously and progressively when maintained in N5 culture medium supplemented with serum proteins, whereas other types of neurons survive. The death of these neurons occurs spontaneously through a mechanism that is dependent on glial cells 24 28 . It could be shown previously that depolarization by elevation of extracellular K⁺ ([K⁺] 30 mM) was efficient in preventing DA cell demise. Note that K⁺-induced depolarization was performed in the presence of 1 μM MK-801 to prevent secondary excitotoxic stress. In some experiments, Ara-C (2 μM) was added to the medium at DIV 1 to 2 after plating to inhibit proliferation of non-neuronal cells (astrocytes, microglia) as well in the presence of 1 μM MK-801 to prevent secondary excitotoxic stress 28 .

Mesencephalic cultures were treated either with 1 μM MPP⁺ at DIV 4 and DIV 5 as previously described 29 , 10 μg/ml low-endotoxin recombinant human Hsp60 dissolved in PBS (hHsp60; Loke Diagnostics ApS, Risskov, Denmark) at DIV 2 to DIV 5 or in combination of both. The hHsp60 contained < 2 endotoxic units of LPS/mg of protein as determined by limulus amoebocyte lysate assay (BioWhittaker, Cologne, Germany). Since hHsp60 has a much weaker effect than MPP⁺ on cell death, we decide to prolonged treatment of the cell cultures compared to MPP⁺ treatment. At DIV 5, medium including MPP⁺ and hHsp60 was removed and cultures were left to recover until DIV 10 in control medium. Trypsin proteolysis (Gibco, Saint Aubin, France) or pre-treatment by boiling of hHsp60 were used to control for Hsp60-mediated effects, as well as additional treatment with polymyxin B (10 μg/ml, PMBS, Sigma-Aldrich, Lyon, France) as a specific LPS inhibitor to control for LPS effects.

Almost pure microglial cultures were obtained using a technique of high-yield isolation of microglia by mild trypsinization 30 . Briefly, neuronal/glial mesencephalic cultures were prepared as described previously except for the culture medium, which was DMEM/F12 nutrient mixture (DMEM/F12; Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS (endotoxin level of the used FCS < 10 EU/ml). After DIV 14, the cultures were washed for 1 minute with DMEM/F12 to eliminate serum and then incubated with a trypsin/EDTA solution (0.25% trypsin, 1 mm EDTA in HBSS; Invitrogen, Cergy Pontoise, France) diluted 1:4 in DMEM/F12 for half to one hour at 37°C until the upper layer (mainly constituted by neurons/astrocytes) was detached. The medium containing the layer of detached cells was aspirated and the highly enriched microglial cell population (98% of pure microglial cells) that remained attached to the bottom of the well was exposed to 500 μl of DMEM/F12 with 10% FCS to allow trypsin inactivation. The cells were treated with low-endotoxin recombinant human Hsp60 (hHsp60) 10 μg/ml and contained < 2 endotoxic units of LPS/mg of protein as determined by limulus amoebocyte lysate assay (BioWhittaker, Cologne, Germany) or LPS 0.1 μg/ml (Sigma-Aldrich, Lyon, France, strain 055:B5), both dissolved in PBS.

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma-Aldrich, Lyon, France) was added to the cells at a final concentration of 0.25 mg/ml and incubated for 1 hour 31 32 33 . Thereafter, the cells were lysed and quantified using a microplate reader (MultiskanReader, ThermoLabsystems, Egelsbach, Germany) at a wavelength of 570 nm. Cell death and cell lysis was based on lactate dehydrogenase (LDH) activity released into the supernatant. LDH activity was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Lyon, France) according to the manufacturer’s protocol.

For Western blotting, cellular protein was isolated using the lysis buffer M-PER Mammalian Protein Extraction Reagent according to the manual (Pierce, Perbio, Brebières, France). Twenty micrograms total protein/lane was loaded onto a reducing 12% SDS-PAGE and electroblotted onto nitrocellulose membranes. After blotting, the internal protein loading controls were prepared according to the manual of MEM Code, Pierce (Perbio, Brebières, France). Afterwards, the membranes were blocked and incubated for 24 hours at 4˚C with a monoclonal mouse anti-Hsp60 antibody (0.5 μg/ml; R&D Systems,Wiesbaden, Germany) and for the loading control with an anti-beta-actin polyclonal rabbit antibody (1:2,000, Sigma-Aldrich, Lyon, France), respectively. For detection, the nitrocellulose membranes were washed and incubated with a HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; goat-anti-mouse or goat-anti-rabbit both 1:50,000). After AB-incubation membranes were incubated in Super Signal Ultra substrate working solution (Perbio, Brebières, France) and exposed to an autoradiographic film (Mat Plus DG Film, Kodak, Maisons-Alfort, France). Afterwards, Western blot signals were analyzed by densitometry using the NIH Image software Scion, and the Hsp60/α-actin ratio was calculated.

All cell cultures were fixed for 15 minutes at room temperature with formaldehyde (4% in PBS) and washed three times with PBS. For primary mesencephalic cell cultures, the survival of DA neurons was determined by tyrosine hydroxylase (TH) immunocytochemistry. The cultures exposed first for 24 hours at 4°C to a rabbit-polyclonal anti-TH antibody (Pel-Freez Biologicals, Rogers, AR, USA) diluted 1:1,000 in PBS containing 0.2% Triton X-100, were then incubated for one hour at room temperature with an anti-rabbit IgG Alexa488 conjugate (1:500; Sigma-Aldrich/RBI, Lyon, France). In order to identify the total number of neurons in the cultures, cultures were incubated for 48 hours at 4°C with a mouse anti-neuronal Nuclei (NeuN) biotin conjugated monoclonal antibody (Chemicon International, Limburg, Germany) and then incubated with a streptavidin-Cy3-antibody (1:1,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature. Mesencephalic cultures contained between 1 and 3% TH⁺ cells at the time of plating. To quantify the cells, the entire TH⁺ and/or NeuN⁺ neurons of the used wells were counted.

In the primary microglial cultures, microglial cells were identified with a rat anti-CD11b Ig (MAC-1; Serotec, Oxfordshire, UK; 1:100 in PBS, 24 hours at 4°C) and revealed using an anti-rabbit IgG Alexa488 conjugate (1:500; Sigma-Aldrich/RBI, Lyon, France). Note that Triton X-100 was not used for the detection of microglial cell surface markers. A polyclonal rabbit beta-tubulin antibody was used (Cell Signaling, Leiden, Netherlands; 1: 200 in PBS containing 0.2% Triton X-100, 24 hours at 4°C) to detect cytoskeletal structures and revealed using an anti-rabbit IgG Alexa488 conjugate (1:500; Sigma-Aldrich/RBI, Lyon, France).

For staining double-stranded DNA, we used PBS containing 10 μg/ml 4’6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Lyon, France). Illustrations of microglial cells are presented as inverted images. For the confocal images, a Leica SP1 confocal microscope was used (Leica, Wetzlar, Germany).

For binding studies, labeled or unlabeled hHsp60 and BSA (Sigma-Aldrich, Lyon, France) were used. The labeled proteins were conjugated to Alexa 647 using the protein labeling kits from Molecular Probes, Saint Aubin, France, according to the manufacturer’s protocol. For binding of hHsp60 to primary microglia 1 × 106 cells were incubated on ice with either 10 μg/ml Alexa 647-labeled hHsp60 or BSA. To exclude an unspecific effect of labeled proteins, cells were also treated with unlabeled proteins (hHsp60 or BSA). Cells that obtained unlabeled hHsp60 were treated with 30 μl of Cohn II fraction (Sigma-Aldrich, Lyon, France) and stained with Hsp60-specific antibody (clone LK-1, 1:100 in PBS), TRITC-labeled goat anti-mouse secondary antibody (1:400 in PBS) and DAPI (1:1,000 in PBS). After staining cells were fixed in PBS/1% paraformaldehyde (PFA), centrifuged onto glass slides, and covered with anti-FADE solution (BiomedDia, Zweibruechen, Germany).

The estimation of protein concentration was performed by the Bradford assay and verified by SDS-PAGE followed by silver staining. Western blot for Hsp60 quantification was made with a specific anti-Hsp60 monoclonal antibody (SPA-810, StressGen, San Diego, CA, USA).

1 to 2 × 10⁶ microglial cells were washed with PBS, detached and incubated with different concentrations of hHsp60 (1 to 50 μg/ml) on ice for 30 minutes. Cells were washed with PBS and Fc receptors of cells were blocked with 30 μl of Cohn II fraction (10 mg/ml, Sigma-Aldrich, Lyon, France) prior to staining with a specific antibody against Hsp60 (SPA-810; StressGen, San Diego, CA, USA). To avoid an internalization of cell surface-bound Hsp60, the incubation of microglial cells with Hsp60 was done on ice. Cells were then washed and a phycoerythrin-labeled goat-anti mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was added.

The supernatant of SH-SY5Y cell cultures was collected at different time points, centrifuged and immediately frozen at -80°C. The detection of Hsp60 was carried out by using a quantitative sandwich immunoassay (Hsp60-ELISA-Kit, StressGen, San Diego, CA, USA) according to the manufacturer’s protocol. Release of microglial cytokines (IL-1β, IL-6 and TNF-α) was measured at different time points (1, 2, 4, 6 and 24 hours) after addition of 10 μg/ml hHsp60.

In addition, microglial cells were treated with hHsp60 that was left untreated, boiled, trypsin-inactivated or pre-incubated with either a specific Hsp60-antibody (Clone 4B9; Dianova, Hamburg, Germany) or an isotypic IgG2-antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany). As a positive control for cytokine release, primary microglial cells were incubated with only LPS (0.1 μg/ml), a prototypical inflammogen known to activate microglia through TLR4 stimulation 34 35 .

Cytokine release into the supernatant was measured employing the DuoSet ELISA Development System mouse IL-1β, IL-6 and TNF-α (R&D Systems, Wiesbaden, Germany). ELISAs were performed according to the manufacturer’s protocol.

Accumulated nitrite, a stable oxidation product of NO, was measured using Griess reagents 36 . Fifty microliters of primary microglial cell supernatants were transferred to a 96-well microtiter plate and 50 μl of solution 1 (1% sulfanilamide in 5% phosphoric acid) were added. After 10 minutes incubation in the dark, 50 μl of solution 2 (0.1% naphthylethylenediamine dihydrochloride) were added and incubated for 10 more minutes in the dark. The absorbance was measured at 450 nm using a plate reader (ELISA-Reader Infinite® 200 series, Tecan, Crailsheim, Germany).

All in vitro experiments were performed in at least three independent experiments, with a minimum of three wells per experimental condition. We confirm that there is no duplication of data presentation in the text. Data are expressed as the percent of corresponding control values. The normalization to control was used because of the high variance due to the different preparations of primary cell cultures. Each data point represents mean ± SEM. Multiple comparisons against a single reference group were performed by one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s test. When all pairwise comparisons were carried out, one-way ANOVA was followed by a post hoc Student-Newman-Keuls test. For the human qPCR experiments, a Spearman correlation test between Hsp60 and TH mRNA expression was calculated. The null hypothesis was rejected at an α risk of 5%.

We postulated that injury of DA neurons could result in the release of Hsp60, causing activation of microglia cells. To test this hypothesis, we first examined the dynamic changes of Hsp60 mRNA expression in the mesencephalon and the striatum of C57/Bl6 mice intoxicated with MPTP. Compared to saline-treated animals, Hsp60 mRNA expression was up-regulated in the mesencephalon and striatum of MPTP-exposed mice, the striatal response being more sustained in time (Figure  1A). Thus, this suggests that MPTP-induced mitochondrial stress in nigral DA neurons can trigger an Hsp60 response in vivo. To confront these results with the human pathology, we further assessed the expression level of Hsp60 transcripts in human post-mortem SN specimen by qRT-PCR. We found a significant increase in the Hsp60/TH ratio (to correct for DA cell loss in PD) in PD patients (n = 5) compared to age-matched control subjects (n = 5) (Figure  1B) suggesting that the Hsp60 stress response may play a role in the pathomechanism of PD.

To further investigate a possible link between the neuronal Hsp60 response and PD-related cellular stress, we treated the human DA cell line SH-SY5Y with the mitochondrial toxin MPP⁺ and first assessed Hsp60 expression. As shown in Figure  2A, exposure of DA cells to MPP⁺ induced a time-dependent up-regulation of Hsp60 expression as assayed by immunoblot analysis on whole cell lysates. Up-regulation was first noticed 12 hours after intoxication and continuously increased up to 48 hours. The level of Hsp60 was not only increased in the intracellular compartment but also in the cell culture supernatant (Figure  2B), suggesting that cellular damage induced by mitochondrial alteration can stimulate the release of stress-related factors from diseased DA neurons. Importantly, increased extracellular Hsp60 observed at 12 and 48 hours following MPP⁺ treatment was not due to permeabilization of cell membranes as the release of LDH (a determinant of cell membrane integrity) was only significant after 24 hours of treatment (Figure  2C). By contrast, decreased mitochondrial cell function evidenced via the MTT assay 37 was detected earlier (six hours) after MPP⁺ treatment and concomitant with the rise in extracellular Hsp60. Altogether, these results indicate that MPP⁺ treatment of SH-SY5Y cells compromised their function and stimulated the release of Hsp60 well before membrane rupture and death, suggesting the existence of an active Hsp60 secretory mechanism when DA neurons are in a suffering/damaged state.

We next addressed the question whether Hsp60 influences neuronal cell death and treated primary mesencephalic cultures with either 1 μM MPP⁺ at DIV 4 and DIV 5, 10 μg/ml hHsp60 at DIV 2 to 5, or a combination of both. At DIV 5, medium including MPP⁺ and hHsp60 was removed and cultures were left to recover until DIV 10 in control medium. Under these experimental conditions, hHsp60 treatment decreased TH⁺ neurons to 68.3% ± 2.6% compared to untreated cultures and the overall population of neurons (NeuN⁺ cells) of these cultures to 74.4% ± 0.7% (Figure  3A). Treatment with MPP⁺ decreased the number of TH⁺ cells to 50.3% ± 1.4%, but did not affect the number of NeuN⁺ cells (98.6% ± 0.7%). Primary mesencephalic cultures contain only 1 to 3% of DA neurons (TH⁺) 38 . These neurons are selectively killed by MPP⁺ at the dosage used, leaving intact the remaining neuronal populations. Thus, the number of NeuN⁺ positive neurons was not significantly affected by MPP⁺ treatment.

Interestingly, co-treatment with hHsp60 and MPP⁺ led to a further loss of TH⁺ neurons in comparison to MPP⁺ or hHsp60 treatment alone (MPP⁺: 50.3% ± 1.4%, hHsp60: 68.3% ± 2.6 co-treatment of MPP⁺/hHsp60: 29.4% ± 1.3%), but exerted no additional effect on NeuN⁺ cells (co-treatment of MPP⁺/hHsp60: 73.6% ± 1.3%, MPP⁺: 98.6% ± 0.7%, hHsp60: 74.4% ± 0.7%) (Figure  3A).

To determine whether neuronal cell death mediated by Hsp60 required glial cell activation, we inhibited the proliferation of glial cells with the anti-mitotic agent AraC (2 μM) applied at DIV 1 for 48 hours 28 . The elimination of glial cells from the cultures curtailed neuronal cell death induced by Hsp60 (Figure  3B). Besides, hHsp60-dependent neurotoxic effects were almost abrogated when hHsp60 was previously denatured by trypsinization (TH⁺: 94.3% ± 1.2%), but not when cultures were exposed to both hHsp60 and the specific LPS antagonist PMBS (TH⁺: 66.3% ± 2.1%), suggesting that the effects of hHsp60 occurred by a mechanism unrelated to that elicited by LPS (Figure  3B).

A direct biological effect of Hsp60 on microglia requires the binding to specific receptors. Therefore, to determine whether Hsp60 could provoke microglial cell-activating properties similar to those previously reported for macrophages 39 40 , we performed in vitro stimulation of primary microglial cell cultures with recombinant hHsp60. For comparative purposes, control cultures were exposed to LPS. Since Hsp60 is thought to induce a pro-inflammatory response through binding to the LPS receptor TLR4 41 , we first analyzed the binding properties of hHsp60 on murine microglia. Incubation of microglial cells with Alexa647-conjugated hHsp60, but not with Alexa647-conjugated BSA used as a negative control, revealed a punctuated distribution of bound hHsp60 suggesting the presence of cell surface receptors that binds to the protein (Figure  4A). Fluorescence-activated cell sorting (FACS) analysis of cells incubated with increasing concentrations of hHsp60 indicated a non linear and saturable binding of hHsp60 to microglia, thus further supporting the view that microglial cells express specific Hsp60 receptors (Figure  4B).

Subsequently, microglial cells were treated with hHsp60 at a concentration of 10 μg/ml. After 1, 2, 4, 6 and 24 hours respectively, hHsp60-exposed cells showed no morphological alterations (that is enlargement of cell bodies; data not shown). Furthermore, they did not release significantly increased amounts of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) or NO compared to the control condition (Figure  4C). In the LPS-treated positive control (0.1 μg/ml), significant values of IL-6 and TNF-α could be shown after 6 and 24 hours respectively for NO. However, a significant increase of IL-1β could not be shown in the LPS-treated group. This may be due to concentrations of LPS used being too low and/or the time of measurement being too early (six hours) to sufficiently stimulate IL-1β release.

In addition, microglial cells were exposed to different hHsp60 preparations (native hHsp60, hHsp60 denatured by trypsinization or boiling and hHsp60 pre-incubated with Hsp60-specific antibody or isotype control antibody). After 12 and 24 hours respectively, hHsp60-exposed cells did not release significantly increased amounts of pro-inflammatory cytokines (IL-6 and TNF-α) compared to the control condition. (Figure  4D). Taken together, these observations strongly suggest that microglia express cell surface receptors to interact with Hsp60. Microglia, however, do not react to Hsp60 with the production of measurable amounts of classical pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α or NO release.