Bindarit

Bindarit, Inhibitor of CCL2 Synthesis, Protects Neurons Against
Amyloid-ti-Induced Toxicity

Cinzia Severinia,∗, Pamela Petrocchi Passerib, MariaTeresa Ciottib, Fulvio Florenzanoc,
Roberta Possentib,d , Cristina Zonab,d , Anna Di Matteoe , Angelo Guglielmottie , Pietro Calissanoc, Joel Pachterf and Delio Mercantib
aInstitute of Translational Pharmacology, CNR, Rome, Italy
bInstitute of Cell Biology and Neurobiology, CNR, IRCCS Fondazione Santa Lucia, Rome, Italy
cEuropean Brain Research Institute, IRCCS Fondazione Santa Lucia, Rome, Italy
dDepartment of Neuroscience, University of Rome “Tor Vergata”, Rome, Italy
eAngelini Research Center, Piazzale della Stazione, Pomezia, Italy
fDepartment of Cell Biology, University of Connecticut, Health Centre, Farmington, CT, USA

Accepted 7 July 2013

Abstract. One of the hallmarks of Alzheimer’s disease (AD), the most common age-related neurodegenerative pathology, is the abnormal extracellular deposition of neurotoxic amyloid-ti (Ati) peptides that accumulate in senile plaques. Ati aggregates are toxic to neurons and are thought to contribute to neuronal loss. Evidence indicates that inflammation is involved in the pathophysiology of AD, and activation of glial cells by a variety of factors, including Ati , appears to be a central event. Among molecules produced during inflammation associated with neuronal death, CCL2, also known as monocyte chemotactic protein-1 (MCP-1), seems to be particularly important. Indeed, CCL2 levels are higher in the cerebrospinal fluid of patients with AD than in controls. In the present study, we demonstrated the protective effect of bindarit (which inhibits CCL2 synthesis) against both Ati 25-35 and Ati 1-42 -induced toxicity in primary mixed neural cultures. Bindarit (30–500 tiM) reversed cell death induced by Ati in a dose-dependent manner and reduced the transcription and release of CCL2 by astrocytes after Ati treatment, as revealed by qRT-PCR, ELISA, and immunofluorescence staining. Astroglial activation and CCL2 release was induced by ATP released by damaged neurons through interaction with P2X7 receptors present on astrocyte surface. CCL2, interacting with its cognate receptor CCR2, present on neuron surface, strongly contributes to the toxic activity of Ati . Bindarit was able to disconnect this neuro–glial interaction. Our results demonstrate the ability of bindarit to inhibit Ati-induced neuronal death and suggest the potential role of CCL2 inhibitors in the treatment of neuroinflammatory/neurodegenerative diseases.

Keywords: Alzheimer’s disease, amyloid-ti toxicity, bindarit, CCL2, neuroinflammation

INTRODUCTION

Alzheimer’s disease (AD) is the most common age-related neurodegenerative pathology, clinically

∗ Correspondence to: Cinzia Severini, CNR, Institute of Trans- lational Pharmacology, Via del Fosso del Cavaliere, 100, 00133 Rome, Italy. Tel.: +39 6 501703234; Fax: +39 6 501703311; E-mail: [email protected].
characterized by progressive impairment of memory and cognition. One of the hallmarks of AD is the abnor- mal extracellular deposition of neurotoxic amyloid-ti (Ati) peptides, products of the amyloid-ti protein pre- cursor (AtiPP), which accumulate in senile plaques [1]. Senile plaques are intimately surrounded by mor- phologically abnormal axons and dendrites and are infiltrated by astrocytes and activated microglia in and around their central amyloid core [2]. Previous

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studies suggested that Ati aggregates are toxic to neu- rons and are thought to contribute to neuronal loss in AD development.
Increasing evidence indicates that inflammatory processes are involved in the pathophysiology of AD [3, 4]. Neuroinflammation represents one of the primary immune processes in response to chronic neu- rodegenerative conditions, like AD, and serves to both “clean up” the lesion and limit the area. Indeed, a cen- tral event in this pathology appears to be the activation of glial cells by a variety of factors, including Ati [5].
Chemokines are a group of cytokines originally identified as factors regulating the migration of leuko- cytes in inflammatory and immune responses [6]. Whileithasbeenreportedthatchemokinesexertphysi- ological action in the healthy brain [7], they have been shown to be produced in response to various patho- logical conditions including multiple sclerosis, brain ischemia and AD [8, 9]. Indeed, several chemokines and chemokine receptors have been found to be upregulated in the AD brain [10, 11]. Among these chemokines, CCL2, also known as monocyte chemo- tactic protein-1 (MCP-1), has been found to be higher in the cerebrospinal fluid of patients with AD than in controls, suggesting it might be one of the key molecules in this pathology [12–15].
Moreover, since CCL2 was found immunohisto- chemically in mature, but not in immature, senile plaques and in reactive microglia of brain tissues from patients with AD, it has been suggested that CCL2-related inflammatory events contribute to the maturation of senile plaques [16]. Recent studies including patients with mild cognitive impairment (MCI), a group at high risk to develop Alzheimer- type dementia over time [17], strongly suggest that the inflammatory process may precede the dementia stages [13, 18]. Thus, anti-inflammatory therapeutic intervention at this time may be useful to prevent severe cerebral damage and clinical progression to dementia.
These observations prompted us to investigate the potential protection of CCL2 inhibition against Ati- induced death in an in vitro model of Ati neurotoxicity. The Ati1-42 peptide is the predominant amyloid pep- tide in AD [19]. However, other minor fragments have also been identified, including the highly toxic Ati 25-35 peptide, which represents the neurotoxic domain of the native, full-length peptide Ati 1-42 [20, 21].
The present study tested, in an in vitro model of Ati25-35 and Ati 1-42 induced toxicity, the effect of bindarit, an original indazolic derivative devoid of immunosuppressive effects and with no activity on arachidonic acid metabolism that was shown to have

anti-inflammatory activity in a number of experimen- tal diseases including nephritis, arthritis, pancreatitis, colitis, and autoimmune encephalomyelitis [22–27].
We performed experiments using cortical primary cultures to characterize the potential neuroprotective activity of bindarit, as well as its mechanism of action.

MATERIALS AND METHODS Chemicals
Ati 1-42 , Ati25-35, and the reverse-sequence peptide Ati35-25 were purchased from Sigma (St. Louis, MO, Missouri). Ati peptide stock solutions at a concen- tration of 1.3 mg/ml were prepared in PBS (0.01 M NaH2 PO4, 0.15 M NaCl, pH 7.4), centrifuged before use to remove the large fibrillar aggregates, and used at 20 tiM concentration. All other reagents were also from Sigma, if not specifically reported. Bindarit was synthesized by and obtained from Angelini (Angelini ResearchCenter-ACRAF,Italy).ABindaritstocksolu- tion (100 mM) was prepared in dimethyl sulfoxide (DMSO) and dilutions of the DMSO stock were made in the culture medium.

Cortical mixed cultures of neural cells

All procedures were approved by the Italian Min- istry of Health (Rome, Italy) and performed in compli- ancewiththeguidelinesoftheUSNationalInstitutesof Health and the Italian Ministry of Health (D.L.116/92). Mixed neural cell cultures (CNs), containing both neu- rons and glial cells (astrocytes and microglia) were pre- pared from brains of embryonic day 17–18 (E17/E18) embryos from timed pregnant Wistar rats (Charles River), as previously reported [28]. In brief, cortex was dissected out in Hanks’ balanced salt solution buffered with Hepes and then dissociated via trypsin treatment. Cells were plated at 1 × 106 cells on 3.5-cm dishes pre- coated with poly-L-lysine. After 2 days of culturing in neurobasal medium with B-27 supplement (0.5 mM L- glutamine, 1% antibiotic penicillin/streptomycin), half of the medium was changed every 3–4 days. All experi- mental treatments were performed on 12-day “in vitro” (DIV) cultures in Neurobasal + ½ B27 fresh medium. The culture cell composition was determined using immunocytochemical staining for neurons (NeuN anti- body, (Sigma, 1 : 200), astrocytes (GFAP antibody, 1 : 400, Sigma), and microglia (Iba1 antibody, Abcam
1: 200) with DAPI nuclear staining. Mixed cultures contain about 45% NeuN+ cells, 50% GFAP+ cells, and 4% of Iba1+ cells.

Cell viability

Cell viability was assessed by counting the number of intact nuclei according to the method previ- ously described [29]. Briefly, the culture medium was removed and replaced with 0.5 ml of a detergent containing lysing solution (0.5% ethyl- hexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaCl, 2 mM MgCl2 , in phosphate-buffered saline (PBS) pH 7.4 diluted 1/10). After 2 min, the suspension of cells was collected. Nucleifromoriginallyviablecellswerethenquantified by counting in hemocytometer as the detergent- containing solution selectively dissolves nuclei of dying cells. Nuclei from viable cells thus appear as phase-bright intact circles surrounded by a dark ring. Broken or damaged nuclei were not included in the count.

Assessment of nuclear morphology

Alternatively, cells were fixed in 4% paraformalde- hyde and permeabilized with 0.2% Triton X-100 in Tris HCl 0.1 M pH 7.4 for 5′ and then incubated with Hoechst 33258 (0.25 tig/ml) for 5 min at room tem- perature. After washing with PBS, the percentage of shrunken and condensed nuclei was assessed. Nuclei were then visualized by a Leica fluorescent photomi- croscope and scored by counting 12 microscopic fields per coverslip in two coverslips from four experiments.

RNA purification, reverse transcription, and RNA determination by quantitative RT-PCR

Total RNA was extracted using the TRIzol solution Invitrogen(Carlsbad,CA,USA),accordingtotheman- ufacturer’s instructions. RNA yield and purity were determined by spectrophotometry absorption at 260 and 280 nm.
To obtain cDNA, 2 tig total RNA was reverse tran- scribed in MLV reverse transcription buffer (Promega, Madison, WI) containing the following: 40 tig/ml random primers (Promega, Madison, WI), 1 mM dNTP, 40 u/ml of Recombinant RNasin Ribonuclease Inhibitor (Promega, Madison, WI), and MLV reverse transcriptase (Promega, Madison, WI) in a final vol- ume of 25 til. The reaction was incubated at 37◦C for 60 min, and the resulting cDNA was stored at -20◦C until used for the further analysis. Relative messenger RNA expression was measured with quantitative real time PCR (qRT-PCR) using a 7900HT Fast-Real time PCR System (Applied Biosystems) and SYBR Select

Master Mix fluorescence (Applied Biosystems) was used to quantify relative amplicon amount.
Cycle time (Ct) values for all samples were normal- ized to Tata Binding Protein (TBP), purchased from Invitrogen) using the titiCt formula. Each cDNA sample from treated cells was assayed in triplicate for each point. For each set of primers, a no template control and a no reverse transcriptase control were included. The thermal cycling conditions were: 95◦C,
2min for denaturation, followed by 40 cycles of 95◦C, 15 s, 60◦C, 1 min. Post-amplification dissociation curves were performed to verify the presence of a single amplification product and the absence of genomic DNA contamination. Relative CCL2 gene expression values (after normalization to TBP) were expressed as the percentage of control. The primer sequences used in this study were as follows: for rat CCL2, forward 5′-TGTAGCATCCACGTGCTGTC- 3′, reverse 5′-CCGACTCATTGGGATCATCT-3′; and for rat CCR-2, forward 5′-CACACCCTGT- TTCGCTGTAGGAATG-3′, reverse 5′- TGGCCTG- GTCTAAGTGCATGTCAA-3′.

ELISA CCL2 determination

At 48 h after apoptosis induction, media from con- trol and apoptotic cells was harvested for CCL2 determination. The level of CCL2 was measured with mouse/rat JE/CCL2 commercial enzyme-linked immunoassay kit (Mouse CCL2/JE/MCP-1 Quan- tikine ELISA Kit, R&D Systems) according to the manufacturer’s instructions. The concentration for each sample was calculated by identifying the optical density (O.D.) value on the standard curve and reading the corresponding value on the x-axis. The concentra- tion read from the standard curve was then multiplied by the dilution factor.

Western blotting

For cytoplasmic lysates, neurons were washed twice with ice-cold PBS, lysed in lysis buffer (1% NP40, 50 mM Tris-HCl, pH 8 and 1X protease inhibitor mixture) and centrifuged. Protein concen- tration was measured using a Biorad DC protein assay kit (Bio-Rad) and equivalent amounts of pro- tein (10–30 tig) were separated on 12% or 15% Bis-Tris SDS-PAGE gels (Invitrogen), blocked with 5% milk for 30 min and then incubated overnight with anti CCR2 antibody (Santa Cruz 1 : 500), with anti P2X7 (Sigma 1 : 200) or anti ti-actin (Sigma (1 : 1000). Incubation with anti-rabbit secondary

antibodies peroxidase-coupled was performed for 1 h at room temperature. Immunoreactivity was devel- oped with enhanced chemiluminescence (ECL system; Amersham, Arlington Heights, IL) and visualized by autoradiography. For analysis of the western blot- ting data, densitometric analysis was performed using Image J software.

Immunocytochemistry

After 48 h of treating 10-day CNs cultures, cells were washed in PBS and fixed in 4% (w/v in PBS) paraformaldehyde for 15 min at room temperature. Fixed cells were washed in PBS, pH 7.4, and perme- abilized using 0.1% Triton X100-Tris-HCl, pH 7.4, for 8 min and then treated with primary polyclonal antibody raised against CCL2 (Santa Cruz) or P2X7 receptor (Sigma) at 1 : 200 dilution in PBS at 4◦C overnight. For CCR2 detection, cells were fixed in 4% paraformaldehyde (15 min) and then incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h to permeabilize them and block non-specific protein-protein interactions. Cells were then incubated with the anti CCR2 anti- body (Santa Cruz) at 1 : 200 dilution overnight at +4◦C, then washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (Sigma 1 : 1000) for 30 min at room temperature. Cells were stained with Hoechst 33258 (0.25 tig/ml) for 5 min at room temperature or with NeuN (Sigma, 1 : 200), MAP2 (Cell Signaling, 1 : 200), or GFAP (Sigma, 1 : 400 dilution). For the control of speci- ficity, primary antibody was omitted from staining reactions. Cells were visualized by a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Confocal acquisition settings were identical among the different experimental cases. For production of figures, brightness and contrast of images were adjusted by applying the same values. Final figures were assembled by using Adobe Photo- shop 7 and Adobe Illustrator 10.

Data analysis

Statistical analysis was performed using SPSS 11.0.0 for Windows (SPSS Inc., USA). All results are expressed as mean ± SEM, with n the number of inde- pendentexperiments.Thesignificanceoftheeffectwas performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple compari- son. The significance level was set at p < 0.05 (*) and p < 0.01 (**). RESULTS Effects of bindarit on Aβ-induced toxicity We first assayed cultures of CNs to determine if bindarit could protect against neurotoxicity induced by Ati 25-35 or Ati 1-42 , as previously described [30]. Bindarit was co-applied with Ati 25-35 or with Ati1-42 (20 ti M) and was present in the medium for the dura- tion of Ati exposure (48 h). The concentrations of bindarit used have previously been found to be effec- tive in inhibiting CCL2 production in mouse astrocytes and microglia [27]. As shown in Fig. 1, quantitative analysis revealed that incubation of CNs with 20 ti M Ati 25-35 (Ati) for 48 h caused ∼55% reduction in the number of surviv- ing cells, as compared to control-treated cells (CTR). Bindarit (BIND) (10, 30, 300, and 500 ti M) dose- dependently reversed the Ati25-35 toxicity, inducing 7.5 ± 4.3%, 23.4 ± 1.7%, 34.5 ± 1.7, and 41.6 ± 3.6% increase in cell viability, as compared to (Ati) con- ditions. In additional experiments, CNs were treated with the reverse-sequence peptide Ati35-25, but no increase in cell death occurred with this peptide (data not shown). The same results were obtained using the full- length peptide Ati1-42 . As reported in Fig. 2, 20 tiM Ati1-42 reduced the number of surviving by ∼45%, as compared to CTR, and this effect was suppressed by treating CNs with different doses of bindarit (30, 100, 300, 500 ti M), beginning with the lowest dose of 100 ti M, which yielded a nearly 16% increase. Increas- ing the dose to 300 and 500 ti M resulted in increases in cell survival to approximately 30% and 43%, respec- tively, of CTR value. As previously demonstrated [30] and confirmed by Hoechst staining (Fig. 2), shrunken and condensed nuclei obtained after Ati treatment indi- cate an apoptotic process. Bindarit effect on Aβ1-42 -stimulated CCL2 mRNA Previous studies reported that, in vitro, bindarit selectively inhibited the production of the monocyte chemotactic protein subfamily of CC inflammatory chemokines (MCP-1/CCL2) at the transcriptional level [31]. We therefore performed qRT-PCR stud- ies to measure CCL2 mRNA. CNs were pre-treated with 500 ti M bindarit for 1 h, then cells were incu- bated with ± 20 ti M Ati 1-42 for 4 h in the presence of bindarit. As shown in Fig. 3, stimulation with 20 tiM Ati1-42 produced a significant increase in CCL2 gene expression (∼3-fold), and bindarit treatment Fig. 1. Effect of bindarit (BIND) against Ati 25-35 neurotoxicity in mixed cortical neurons (CNs). CNs (1 × 106 cells/well) were treated at 12 DIV with 20 tiM Ati 25-35, alone or in the presence of increasing concentrations of BIND (10, 30, 300, 500 tiM) and then assayed for cell viability 48 h later. Data from the same concentrations of BIND alone are also shown. Data represent mean (±SEM) from at least five independent experiments run in duplicate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s test for multiple comparisons (**p < 0.01 versus CTR; #p < 0.01 versus Ati treatment). Fig. 2. Protective effect of bindarit on nuclear condensation induced by Ati 1-42 in terms of Hoechst staining. Representative immunofluorescence photomicrographs showing CNs nuclei stained with Hoechst. a) control, b) CNs after 48 h of 20 tiM Ati 1-42 treatment, c) cells exposed to 20 tiM Ati 1-42 for 48 h simultaneously to bindarit (BIND 500 ti M). Histogram shows quantification of live nuclei respect to Hoechst-stained (apoptotic) nuclei for each treatment expressed as percent of control. 12 fields were selected for each treatment from three independent experiments (n = 3). Data represent means (±SEM) and statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s test for multiple comparisons (**p < 0.01 versus CTR; #p < 0.01 versus Ati 1-42 ). Fig. 3. Bindarit effect on CCL2 mRNA. CNs were pretreated with 500 tiM bindarit for 1 h and then cells were incubated with ± Ati 1-42 20 tiM for 4 h in the presence of bindarit. Relative CCL2 mRNA lev- els were determined by qRT-PCR, and effects of bindarit treatment reported as % change compared to control cultures. Data represent mean (±SEM) from two independent experiments run in tripli- cate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s test for multiple comparisons (**p < 0.01 versus CTR; #p < 0.01 versus Ati treatment). suppressed, by 40 to 60%, the induction of CCL2 mRNA. In addition, we tested the effect of bindarit on CCR2 gene expression. However, no significant vari- ations were obtained for this cognate CCL2 receptor (data not shown). Effect of bindarit on CCL2 release Given the elevation of CCL2 mRNA detected in response to treating CNs with Ati peptide, we next measured the CCL2 protein content in the cellular media by ELISA. As shown in Fig. 4, incubation of CNs with 20 tiM Ati 25-35 for 48 h induced a significant increase in CCL2 levels as compared to control con- ditions (CTR). 30, 300 and 500 tiM bindarit (BIND) provoked 25 ± 4.6%, 43.4 ± 2.6%, and 49.6 ± 3.9% reduction in CCL2 production, as compared to Ati alone. CCL2 and CCR2 expression in CNs We examined the expression of CCL2 and its cog- nate receptor CCR2 by the use of rabbit polyclonal anti-CCL2 and anti-CCR2, respectively. Figure 5A demonstrates that CCL2 is mainly present in astro- cytes. A CCL2-immunoreactive signal (green channel) is distributed in the processes of astrocytes that are selectively labeled by antibody to GFAP (red chan- nel). As shown by the merge, there is immunoreactive overlap between astrocytes and CCL2, confirming CCL2 is mainly confined into astrocytes in these mixed neural cell cultures. Figure 5B shows the immunore- active localization of the membrane receptor CCR2. Immunofluorescence staining demonstrates that CCR2 is localized in neurons. A CCR2 immunoreactive signal (red) is distributed in the plasma membrane and cytoplasm in neuronal (MAP2 positive) cells. As shown by the merge panel, coexpression of the two markers is observed, confirming the receptor is present in neuronal cells. The presence of CCR2 in these cell lysates was confirmed by western blot analysis, as shown in the same figure. Effect of recombinant CCL2 and of CCR2 antagonist To support the hypothesis that CCL2 is a media- tor of toxicity in these CNs cultures, we performed direct studies using different concentrations of syn- thetic CCL2 (data not shown). As shown in Fig. 6A, 100 ng/ml CCL2 exerted a significant toxic effect, comparable to that achieved with 20 ti M Ati, lead- ing to ∼40% decrease in the number of surviving cells. As expected, this effect was not antagonized by bindarit treatment (Fig. 6A), which suppresses only CCL2 synthesis, not activity. To further characterize the contribution of CCL2 in the mechanism of Ati neurotoxicity, we used the selective CCR2 antagonist, RS136270 [32]. Quantitative analysis confirmed that incubation of CNs with 20 ti M Ati 25-35 (Ati) for 48 h induced about 45% reduction in the number of surviv- ing cells, as compared to control-treated cells (CTR). As shown in Fig. 6A, RS136270 (RS) (5 tiM) significantly reversed the Ati 25-35 toxicity, inducing 27.3 ± 2.8% increase in cell viability, as compared to treatment with Ati alone. ATP-induced neurotoxicity is inhibited by bindarit: An ATP:CCL2 link in Aβ neurotoxicity Several lines of evidence indicate that Ati may directly activate astrocytes and oligodendrocytes to induce CCL2 production [33]. However, release of chemokines by astrocytes may also be a consequence of the stimulation by factors released by damaged neurons, among which is ATP [34]. To confirm this hypothesis, we investigated the effect of exogenous ATP on viability of mixed neural cell cultures. As previously demonstrated, incubation of CNs with Ati (20 tiM) for 48 h resulted in ∼40% reduction in the number of surviving cells, as compared to control (CTR), and bindarit (500 ti M) reversed this Ati25-35 Fig. 4. Effect of BIND on CCL2 release in CNs. At 12 DIV, CNs (1×106 cells/well) were treated for 48 h with 20 tiM Ati 25-35 , alone or in the presence of increasing concentrations of bindarit (10, 30, 300, 500 tiM) and cell media were collected to measure CCL2 content by ELISA. Quantification of CCL2 was expressed in pg/ml. Data represent mean (±SEM) from at least five independent experiments run in duplicate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s test for multiple comparisons (**p < 0.01 versus CTR; #p < 0.01 versus Ati treatment). toxicity. As shown in Fig. 6B, application of ATP (300 tiM) reduced the number of surviving cells by ∼50%, an effect that was also reversed by bindarit. Involvement of astroglial P2X7 receptor in CCL2 production Since it has been reported that activation of P2X7 purinoreceptors, for which ATP is a ligand, induces CCL2 synthesis in astrocytes cultures [35], we tested whether this receptor was involved in the Ati -mediated CCL2 release. First, we examined the immunoreac- tivity of astrocytes to an antibody against the P2X7 receptor. Immunostaining with this antibody demon- strated that in control CNs, P2X7 receptors are mainly confined to neurons (Fig. 7A). However, after exposure to Ati, we observed an upregulation of P2X7 receptors in some GFAP-positive astrocytes (Fig. 7B). DISCUSSION Increasing evidence suggests that inflammation sig- nificantly contributes to the pathogenesis of AD [9]. In the amyloid hypothesis of this neurodegenerative dis- ease,ithasbeenproposedthattheAti plaquesstimulate a chronic inflammatory reaction to clear this debris [36]. Indeed, the Ati plaques contain dystrophic neu- rites, activated microglia, and reactive astrocytes [3, 37, 38]. Inflammation clearly occurs in pathologically vul- nerable regions of the AD brain, with increased expression of acute phase proteins and proinflamma- tory cytokines, which are hardly evident in the normal brain [5, 39, 40]. There is also growing awareness that monocyte chemoattractant proteins (MCPs), which belong to the beta chemokine family, might play a possible pathogenic role in AD. In this regard, CCL2 (MCP- 1) and its receptor CCR2 have been argued to play an as yet uncharacterized role in this pathology and other neuroinflammatory diseases, contributing to neuronal deathandcerebraldysfunction[14].Itwouldthusstand to reason that anti-inflammatory drugs targeting neu- roinflammatory mechanisms could be useful in either delaying or slowing disease progression. In particular, drugs able to inhibit CCL2 may be of critical impor- tance in resolving the action of this chemokine in the pathophysiology of AD. Bindarit, 2-methyl-2-[[1-(phenylmethyl)-1H- indazol-3-yl]methoxy]propanoic acid, is an original indazolic derivative with anti-inflammatory activ- ity, able to inhibit MCP-1/CCL2, MCP-3/CCL7 and MCP-2/CCL8 synthesis [31], through down- regulation of the NF-ti B pathway [41]. Bindarit has shown clinical efficacy in a broad array of experimen- tal inflammatory, autoimmune, and vascular disorders [23–25, 42, 43], as well as success in recent clinical trials for diabetic nephropathy and lupus nephritis [44], indicating that its beneficial effects are related Fig.5. CCL2andCCR2expressioninCNs.A)ImmunofluorescenceimagesofmixedCNsdisplayingCCL2expression.Permeabilizedcellswere labeled with the selective polyclonal anti CCL2 antibody (green). Cells were conterstained with Hoechst (blue) and with the astrocyte-specific marker glial fibrillary acid protein (GFAP) (red). As shown by the merge, there is immunoreactive overlap between astrocytes and CCL2. B) Immunofluorescence images of mixed CNs displaying CCR2 expression. Permeabilized cells were labeled with the selective polyclonal anti CCR2 antibody. Cells were conterstained with Hoechst and with selective neuronal marker MAPII. As shown by the merge, there is immunoreactive overlap between neurons and CCR2. Three separate experiments gave qualitatively identical results. Scale bar 15 tim. Western blot analysis of CCR2. Immunoreactive signal of CCR2 band (42 kDa) from control cells normalized against ti -actin run in duplicate. Molecular mass markers (in kilodaltons) are shown on the right. to its anti-CCL2 action. Recently, bindarit was also demonstrated to inhibit development and partially suppress the neuroinflammatory condition experi- mental autoimmune encephalomyelitis, a recognized animal model for multiple sclerosis. Specifically, CCL2 expression in both vascular and parenchymal compartments of the CNS were significantly lowered by systemic bindarit treatment, establishing that bindarit can access critical CNS CCL2 depots to execute its therapeutic effect(s) in neuroinflammatory disease [27]. Results of our work indicate that bindarit exerted a concentration-related neuroprotective activity against both Ati 25-35 and Ati1-42 toxicity. Specifically, in cul- tures of mixed cortical neural cells, bindarit reduced Ati-related neurotoxicity in a dose-dependent manner. This effect correlated with CCL2 suppression at both mRNA and protein level. Fig. 6. CNs viability. A) Mixed CNs (1 × 106 cells/well) were treated at 12 DIV with recombinant CCL2 (100 ng/ml) alone or in the presence of BIND (500 tiM), or with the selective CCR2 antagonist RS136270 (RS, 5 tiM), Ati 25-35 (20 tiM), alone or in the presence of RS and then assayed for cell viability 48 h later. B) Effect of Ati 25-35 (20 ti M), ATP (300 ti M), and BzATP (Bz 100 ti M) alone or in the presence of bindarit (500 tiM). Data represent mean (±SEM) from at least four independent experiments run in duplicate. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s test for multiple comparisons (**p < 0.01 versus CTR; #p < 0.01 versus Ati treatment, § p < 0.01 versus ATP treatment). In the brain, different cell types including neurons may produce chemokines, however, the main neural cells producing CCL2 in response to Ati seem to be astrocytes [33, 45]. Consistent with these findings, we demonstrated that CCL2 was produced by astrocytes in response to Ati in mixed neural cell cultures, since we detected the presence of CCL2 in astrocytes by immunochemical studies. The localization of CCR2 within the nervous sys- tem is heavily debated: although CCR2 expression has mainly been described in glial cells, several studies reported constitutive as well as inducible neuronal expression [46]. Previous findings demon- strated that, in the medullary dorsal horn, CCL2 was present in astrocytes, while its preferred receptor, CCR2 was mainly expressed in neurons, indicat- ing that CCL2-CCR2 axis might be involved in the astroglial–neuronal signaling mediating trigemi- nal neuropathic pain [47, 48]. In agreement with these data, our immunofluorescence results demonstrated the prevalent localization of CCR2 receptors in neu- rons, suggesting that CCL2, released by astrocytes, modulates neuronal response interacting with its cog- nate receptor present on neurons. To confirm this hypothesized mechanism of action, we established that CCL2 was responsible for cell tox- icity, since recombinant CCL2 exerted a toxic activity on cortical mixed neural cells and a CCR2 antago- nist prevented the Ati toxicity mediated at least in part by CCL2. Significantly, toxicity due to exogenous CCL2wasnotinhibitedbybindarit,thussupportingthe hypothesis that this drug acts upstream on astrocytes synthesizing and releasing CCL2. The mechanism of action by which Ati activates glial cells is at present unknown, although there are data indicating that Ati itself may activate astrocytes and oligodendrocytes inducing CCL2 production [33, 49, 50]. Despite this possible direct action of Ati on astro- cytes, it cannot be excluded that such release of chemokines by astrocytes may result from glial acti- vation by factors released by damaged neurons, especially ATP [34]. This molecule has been draw- ing attention as a principal mediator in neuro–glial communication [51, 52], and it has been demon- strated that high levels of ATP are released during trauma, ischemia, and other CNS insults [53]. Since an association between AD and ischemic stroke has been established [54], we speculate that ATP could be released by damaged neurons following Ati insult. Recently, it was demonstrated that, in organotypic cor- ticostriatal slice cultures, ATPtiS markedly induced CCL2 secretion via P2 purinoceptors [34]. Results of thepresentworkconfirmedthatATPwasabletoinduce a toxic effect, comparable to that achieved with Ati incubation, and that its activity was antagonized by bindarit. Extracellular ATP exerts biological effects by act- ing on cell surface P2-purinergic receptors, divided into two major subclasses, P2Y receptors, which are G-protein-linked,andP2Xreceptors,whichareligand- gated ion channels [55]. Among P2X receptors, it Fig. 7. P2X7 receptor expression in CNs. A) Immunofluorescence images of mixed CNs in control conditions (CTR) displaying P2X7 receptor expression. Permeabilized cells were labeled with the selective polyclonal anti P2X7 receptor antibody (green). Cells were conterstained with Hoechst (blue) and with the selective neuronal marker NeuN (red). As shown by the merge, there is immunoreactive overlap between neurons and P2X7 protein. Scale bar 25 ti m. Western blot analysis of P2X7 . Immunoreactive signal of P2X7 band (45 kDa) from control cells run in duplicate normalized against ti-actin. Molecular mass markers (in kilodaltons) are shown on the right. B) Immunofluorescence images of mixed CNs after 48 h Ati 25-35 treatment, displaying P2X7 receptor expression. Permeabilized cells were labeled with the selective polyclonal anti P2X7 receptor antibody (red). Cells were conterstained with Hoechst (blue) and with the astrocyte-specific marker glial fibrillary acid protein (GFAP) (green). Colocalization of GFAP and P2X7 is indicated by double staining in MERGE. As shown in this panel, the P2X7 receptor is present in some cortical astrocytes. Three separate experiments gave qualitatively identical results. Scale bar 10 tim. has been reported that, in rat primary cortical neu- rons, activation of the P2X7 receptor by its agonists BzATP or ATP promoted the cleavage of caspases- 8/9/3 and the appearance of cellular markers indicative of apoptosis [56]. Confirming these data, we demon- strated by immunocytochemistry that in control mixed neural cells P2X7 receptors are mainly confined to neurons. Fig. 8. Schematic and simplified illustration of the mechanism pro- posed to mediate bindarit (BIND) activity on Ati toxicity. However, astrocytes express a number of puriner- gic receptors [57] among which P2X7 receptors and their activation by ATP increase CCL2 expression [35]. These results suggest that the activation of astro- cyte P2X7 receptors may be an integral component of the inflammatory response in the brain. Indeed, these receptors play a critical role in the neuroinflammation observedduringthepathogenesisofneurodegenerative diseases since their pharmacological blockade results in amelioration of neuropathology in animal models of these pathologies [58]. Supporting this data, we observed that Ati induced an upregulation of P2X7 receptors in some GFAP-positive astrocytes, in paral- lel with the presence of CCL2 in astrocytes. This result appears in agreement with data reporting that P2X7 receptor expression is upregulated in transgenic mice overexpressing mutant Ati PP and around amyloid plaques, regionally associated with activated microglia and astrocytes [59]. In light of all these collective data, we propose the following mechanism: a) Ati induces neuronal toxicity and this effect is associated with release of ATP by neurons; b) ATP interacts with P2X7 recep- tors overexpressed in astrocytes after Ati treatment; c) astroglial activation provokes CCL2 release; d) CCL2, interacting with its cognate receptor CCR2 present on neuron surface, strongly participates to the toxic activity of Ati; d) bindarit is able to disconnect this circuit, inhibiting CCL2 transcription and production and ameliorating Ati toxicity (Fig. 8). In conclusion, this study demonstrates that bindarit is effective in preventing expression of the pro- inflammatory chemokine, CCL2, induced in vitro by exposure to Ati. 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