The immortalized murine microglial BV-2 cell line [6], was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum (FBS) and 50 µg/ml gentamicin at 37℃. Mouse primary microglial cells were maintained in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco) at 37℃ in a humidified atmosphere with 5% CO₂. All animals and experimental procedures were approved by the Institutional Review Board of Kyungpook National University School of Medicine, and were carried out in accordance with the guidelines in the NIH Guide for the Care and Use of Laboratory Animals. The animals were maintained under temperature- and humidity-controlled conditions with a 12-h light/12-h dark cycle. The mouse primary microglial cultures were prepared using mild trypsinization as previously described, but with minor modifications [7]. In brief, the forebrains of 3~5 day-old C57BL/6 mice were chopped and dissociated through mechanical disruption using a nylon mesh. The cells were seeded into poly-L-lysine-coated flasks. After in vitro culture for 10~14 days, the microglial cells were isolated from the mixed glial cultures using mild trypsinization. The mixed glial cultures were then incubated with a trypsin solution (0.25% trypsin, 1 mM EDTA in Hank's balanced salt solution) diluted 1:4 in phosphate-buffered saline (PBS; 150 mM NaCl, 5 mM phosphate, pH 7.4) containing 1 mM CaCl₂ for 30~60 min. This procedure resulted in the detachment of an upper layer of astrocytes; the microglia remained attached to the bottom of the culture. The detached layer of astrocytes and the remaining microglia were treated with 0.25% trypsin separately and used for subsequent experiments. The purity of the cultures was greater than 95%, as determined by immunocytochemistry using a rabbit polyclonal anti-Iba-1 antibody (1:1000 dilution; Wako) or anti-GFAP antibody (1:1000 dilution).

The production of nitric oxide (NO) was estimated by measuring the amount of nitrite, a stable metabolite of NO. The cells were treated with lipopolysaccharide (LPS from E. coli 055: B5; Sigma) in the presence or absence of the inhibitors for PTP1B [8,9], TC-PTP, SHP2, MEG2, LYP, and RPTPβ. At the end of a 24 h incubation period, 50 µl of the cell culture media was mixed with an equal volume of a Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) in a 96-well microtiter plate. The light absorbance was read at 540 nm and sodium nitrite was used for a standard curve.

Cel l viabi lity was assessed using a modified 3-(4,5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, as previously described [10]. After LPS treatment for 24 h, either in the presence or absence of pharmacological inhibitors, the culture media was aspirated. MTT (0.5 mg/ml in PBS) was added to cells, which were then incubated at 37℃ for 4 h. The resulting formazan crystals were dissolved in DMSO. The absorbance was determined at 570 nm using a microplate reader.

Total RNA was extracted from the brain tissues or treated cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The reverse transcription (RT) was conducted using the Superscript II reverse transcriptase (Invitrogen) and an oligo (dT) primer. Traditional polymerase chain reaction (PCR) amplification was performed using specific primer sets at an annealing temperature of 55~60℃ for 20~30 cycles. PCR was performed using a C1000 Touch Thermal Cycler (Bio-Rad, Richmond, CA, USA). For the PCR product analysis, 10 µl of each PCR reaction was electrophoresed on a 1% agarose gel and detected under ultraviolet light following ethidium bromide staining. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences were designed based on published cDNA sequences (Table 1).

For histochemical analysis, mice were transcardially perfused with saline and whole brains were fixed in 4% paraformaldehyde for 72 h. The fixed brains were incubated in 30% sucrose for 72 h, embedded in Optimal Cutting Temperature (O.C.T.) compound (Tissue-Tek), and then cut into 12-µm-thick sagittal sections. The sections were permeabilized with 0.3% Triton X-100 and blocked with 1% BSA and 5% normal donkey serum for 1 h at room temperature. The brain sections were incubated with primary antibodies (rabbit polyclonal anti-Iba-1 (1:500 dilution) at 4℃ overnight, followed by an incubation for 1 h at room temperature with secondary antibodies (Cy3-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch Laboratories). The anti-fade mounting medium containing DAPI (VECTASHIELD, Vector laboratories) was used for mounting and counterstaining. Tiled images of each section were captured with a CCD color video camera (Olympus D70) through a 63x objective lens attached to a microscope (Olympus BX51).

LPS was administered intraperitoneally (i.p.) to induce neuroinflammation in mice, as previously described [11]. All experiments were carried out on 9~11-week-old male C57BL/6 mice (25~30 g) supplied by Koatech (Pyongtaec, Korea). To evaluate the expression of PTPs in the brain under an inflammatory condition, LPS was injected i.p. at a dose of 5 mg/kg. The brains were collected 48 h after LPS administration. To assess the effect of PTP inhibitors on neuroinflammation, animals were divided into 8 experimental groups. Group 1 was treated with saline and 0.5% DMSO, group 2 was treated with LPS and 0.5% DMSO, and groups 3~8, were treated with LPS and each inhibitor of PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ diluted in saline containing 5% propylene glycol. DMSO was included in the vehicle because PTP inhibitors were dissolved in DMSO. LPS (5 mg/kg) was administered i.p. for a single challenge. Inhibitors or vehicle were administered intracerebroventricularly (i.c.v.). For histological analysis, the mice were anesthetized 48 h after the LPS injection, and then transcardially perfused with saline and then with 4% paraformaldehyde. Microglial activation was assessed using Iba-1 staining. At least 3 animals were used for each experimental group. Immunohistological intensity analysis of Iba-1 staining was performed using Image J software (NIH, Bethesda, MD, USA) as previously described [12]. The image was set with a binary threshold of 50% of the background level, and then the particles were converted to a subthreshold image area with a size of 100 to 1000 pixels, which shows the Iba-1-positive activated cells. To count the Iba-1 positive cells, 5 squares (300 × 300 µm) were placed around the injection site in the subthreshold image of 6 independent sections, and the cells in the 5 squares were counted and statistically analyzed. Then, the counted cell number was expressed as cell number per square millimeter (cells/mm²).

All data are presented as mean±SE from 3 or more independent experiments, unless stated otherwise. The statistical comparisons between the different treatments were performed using either a Student's t-test or a one-way ANOVA in GraphPad PRISM (Graphpad Software. San Diego, CA, USA) and Excel. To determine the statistical significance of more than 2 groups, the values were compared using a one-way ANOVA followed by a Tukey's multiple comparison test (parametric test) or a one-way ANOVA with a Dunn's test (non-parametric test). For the comparison of 3 groups, the unpaired two-tailed Student's t-test was used, followed by a Mann-Whitney correction for nonparametric data.

To investigate the role of PTPs in the mouse brain under an inflammatory condition, we first analyzed the mRNA expression of several PTPs. Five non-receptor type of PTPs (subtype NT1-NT4) and 1 receptor type of PTPs were selected in this study (Table 2) because these PTPs were well studied and the specific inhibitors have been developed. For an animal model of neuroinflammation, we used LPS-injected mice. Whole brains were collected 48 h after an i.p. injection of LPS (5 mg/kg). The gene expression levels of PTP1B (also known as PTPN1), TC-PTP (also known as PTPN2, a phosphatase that is highly homologous to PTP1B), SHP2 (also known as PTPN11), MEG2 (also known as PTPN9), LYP (also known as PTPN22) and RPTPβ (also known as RPTPz) were assessed by RT-PCR using gene-specific primers. We found that all PTPs were expressed in the mouse brain. PTP1B expression was increased after 48 h (Fig. 1), which is consistent with our previous report [4]. SHP2 and LYP were slightly increased compared to vehicle control, but LPS stimulation did not alter the expression of the other PTPs (Fig. 1).

Because microglia are the resident immune cells in the CNS and participate in the initiation and propagation of an inflammatory response, we examined PTPs expression in primary microglia using RT-PCR. PTP1B, TC-PTP, and LYP, but not MEG2, SHP2, or RPTPβ mRNA levels were increased after 24 h of LPS stimulation (100 ng/ml) (Fig. 2). Similarly, PTP1B, TC-PTP, and LYP mRNA were upregulated after LPS and IFNγ stimulation in primary astrocytes (Fig. 2). Taken together, our results indicate that inflammatory stimuli upregulate several PTPs in glia.

Microglia are the resident immune cells of the brain, and are part of an important defense mechanism. However, uncontrolled inflammatory activation of microglia has been observed in many neurodegenerative diseases [2]. Activated microglia play a central role in neuroinflammation by secreting various neurotoxic factors, such as nitric oxide (NO). Next, we explored the possibility that PTP inhibitors could reduce microglial activation. To test this hypothesis, we used specific inhibitors of PTPs that we previously developed [9,13,14,15,16]. PTPs share a conserved catalytic domain for phosphatase enzyme activity. Nevertheless, specific inhibitors for PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ have been shown to be highly specific [8,9]. Table 2 shows the chemical name and the target protein of the inhibitors used in our study. The levels of NO production were determined in the culture media of the LPS-treated microglial cells that were co-treated with inhibitors at different concentrations. LPS-induced NO levels were decreased by inhibitors for PTP1B, TC-PTP, SHP2, LYP, and RPTPβ in a dose-dependent manner. The IC₅₀ value of each PTP inhibitor is listed in Table 3. PTP inhibitors themselves did not alter the basal levels of NO production. No significant cytotoxicity was observed for any of the PTP inhibitors, except that for SHP2 at concentrations of 10 µM, as determined in a MTT assay (Fig. 3 right). It will be interesting to evaluate the effects of these PTP inhibitors on microglial iNOS expression and neuronal loss in LPS-injected mice in the future.

Finally, we examined whether the PTPs inhibitors influence microglial activation in vivo , which is a hallmark of neuroinflammation [17,18,19,20,21]. The brain tissues were collected and stained with anti-Iba-1 antibody, which is a microglia marker, to evaluate the intensity of Iba-1 staining and microglial morphological changes 48 h after i.p. injections of LPS (Fig. 4A). LPS significantly increased the number of Iba-1-positive activated microglia (Fig. 4D). The inhibition of PTP activity via i.c.v. injection of PTP1B, and RPTPβ inhibitors significantly reduced LPS-induced microglial activation in the hippocampus and cortex (Fig. 4C and D). TC-PTP inhibitors reduced LPS-induced microglial activation in the cortex, and SHP2 reduced microglial activation in the hippocampus. As shown in Fig. 4B, microglial morphological change was observed after LPS injection. The number of activated microglia was quantified after delivery of the PTP inhibitors. Taken together, our results show that inhibiting PTP1B, TC-PTP, SHP2, and RPTPβ activity under inflammatory conditions attenuated microglial inflammatory activation in vitro and in vivo.