MNPs@SiO2(RITC) and silica NPs
MNPs@SiO2(RITC) composed of a 9-nm cobalt ferrite core (CoFe2O3) and an RITC-encompassed silica shell [16] were purchased from Biterials (Seoul, South Korea). The MNPs@SiO2(RITC) were determined to be 50 nm in diameter using transmission electron microscopy (TEM) (Supplementary Fig. 1), and the MNPs@SiO2(RITC) had a zeta potential between –40 and –30 mV [16, 17]. X-ray diffraction (XRD) using a High-Power X-Ray Diffractometer (Ultima III, Rigaku, Japan) confirmed the structure of the MNPs@SiO2(RITC), which showed specific patterns of CoFe2O4: (220) at 30°, (311) at 36°, (400) at 44°, (511) at 57°, and (440) at 64°. The broad peak between 20° and 40° indicated amorphous silica beads (Supplementary Fig. 2). The biological changes of HEK293 cells were induced by the shell (silica) rather than the cobalt ferrite core of MNPs@SiO2(RITC) [8, 18-22]. MNPs@SiO2(RITC) uptake was plateaued at 0.1 μg/μl MNPs@SiO2(RITC) treatment and cell viabilities of BV2 cell line and primary rat microglia were ~80% reduced at 1.0 μg/μl MNPs@SiO2(RITC) treatment (data not shown). Thus, the MNPs@SiO2(RITC) treatment dose to microglia was determined as 0.01 to 0.1 µg/µl in this study.
Primary cell isolation and cell culture
Primary cortical, dopaminergic neurons, and microglia were isolated from Sprague–Dawley rats (1 day pups). Briefly, rat brains were separated into cortex and midbrain and homogenized in Minimum Essential Medium (MEM, Gibco, NY, USA). The cell fractions were cultured in dishes containing MEM supplemented with 10% fetal bovine serum (Gibco, NY, USA), 100 units/ml penicillin, and 100 ng/µl streptomycin (Gibco, NY, USA) in a humidified 5% CO2 chamber at 37°C. Primary rat microglia were isolated using the “shaking off” method [23]. The purity of the primary neuronal cells was verified based on specific marker protein expression: neuronal nuclei (NeuN) for cortical neuronal cells and tyrosine hydrolase (TH) for dopaminergic neuronal cells. The purity of the primary rat microglia was verified by flow cytometry after staining with a specific antibody (CD11b/c, integrin αM; clone OX-42, Santa Cruz, CA, USA) (Supplementary Fig. 3).
Immunocytochemistry
After MNPs@SiO2(RITC) treatment or coculture on cover slips, cells were fixed in Cytofix buffer (BD Biosciences, CA, USA) at 4°C for 30 min. PBS containing 2% bovine serum albumin and 0.1% Triton-X100 (Sigma-Aldrich, MO, USA) was used for blocking the cover slips. The cells were then incubated with anti-Iba1 goat polyclonal antibody (Novus biologicals, CO, USA), anti-d-serine rabbit polyclonal antibody (1:200, Abcam, Cambridge, UK), anti-vesicle-associated membrane protein 2 (VAMP2) mouse polyclonal antibody (1:200, GeneTex, CA, USA), anti- neuropeptide Y (NPY) mouse polyclonal antibody (1:200, GeneTex, CA, USA), anti-ubiquitin rabbit polyclonal antibody (1:200, Santa Cruz, CA, USA), and anti-alpha synuclein monoclonal antibody (1:200, BD Biosciences, CA, USA) diluted in blocking buffer at 4°C for 12 h. After three washes with PBS containing 0.1% Triton X-100, they were incubated with Alexa Fluor 488-, 547-, and 647-conjugated secondary antibodies (1:200, Invitrogen, CA, USA) at room temperature (RT) for 1 h. The labeled cells were washed thrice with PBS containing 0.1% Triton X-100 and incubated with PBS containing 10 µg/ml Hoechst 33342 at RT for 10 min. After three washes with PBS, the cover slips were mounted onto slides using Prolong Gold Antifade mounting medium (Molecular Probes, OR, USA). Fluorescence images were taken using a slide scanner (Axioscan Z1, Carl Zeiss Microscopy GmbH, Jena, Germany) or by high-resolution confocal microscopy (Nikon A1, Nikon, Tokyo, Japan) at the 3D immune system imaging core facility of Ajou University. To quantify inclusion bodies, total cells were counted using ImageJ (NIH Image, Bethesda, MD) and the frequency of aggresome-containing cells was calculated.
TEM
To analyze ultrastructural changes, Karnovsky’s fixative (Sigma-Aldrich, MO, USA) was used for MNPs@SiO2(RITC)-treated BV2 cells fixation at 4°C for 12 h. After fixation, samples were washed with 0.1 M cacodylate buffer (pH 7.4). Cacodylate buffer (0.1 M) with osmium tetroxide [1% (v/v), Polysciences, PA, USA] was used for post-fixation at RT for 2 h. The samples were dehydrated with graded ethanol solutions (50%–100%), infiltrated with propylene oxide, and embedded in Epon (Polysciences, PA, USA). The samples were incubated at 35°C for 6 h, 45°C for 12 h, and 60°C for 24 h. The blocks were sectioned using an ultramicrotome (Reichert-Jung, Bayreuth, Germany). The sections were double-stained with 6% uranyl acetate (Electron Microscopy Sciences, PA, USA) for 20 min and lead citric acid for 10 min (Thermo Fisher Scientific, CA, USA) for contrast. Images were obtained using a SIGMA500 transmission electron microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at the 3D immune system imaging core facility of Ajou University. Particle number, vesicle size, mitochondria number, and size were analyzed using the Zen blue 2.3 (Carl Zeiss Microscopy GmbH, Jena, Germany) image analysis module.
Inductively coupled plasma mass spectrometry (ICP-MS)
MNPs@SiO2(RITC) in BV2 were quantified by ICP-MS (7700, Agilent, Japan) as reported [16]. Briefly, BV2 cells were incubated with 0, 0.01, or 0.1 µg/µl MNPs@SiO2(RITC) for 12 h, and 5 × 106 cells were collected. The treated cells and MNPs@SiO2(RITC) were digested and completely dissolved in concentrated aqua regia and hydrogen fluoride (HF). CoFe2O4, which is the core of the MNPs@SiO2(RITC), contains approximately 10–18 mmol cobalt ions. Thus, the number of MNPs@SiO2(RITC) in each BV2 cell was calculated.
RNA-seq
To analyze transcriptome of 0.01, or 0.1 µg/µl MNPs@SiO2(RITC) treated BV2 cells, total RNA was extracted using a TruSeq Stranded Total RNA Library Prep Kit (Illumina, CA, USA). An Agilent RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany) was used for analyzing RNA quality. mRNA was enriched using Magnetic beads conjugated with oligo(dT). Double-stranded cDNA was synthesized with purified mRNA after fragmentation. Sequencing adapters using a TruSeq RNA Sample Prep Kit (Illumina, CA, USA) was used for modification, including end-repair and poly(A) addition and connection, of the cDNA. The samples were isolated using Blue Pippin (Sage Science, MA, USA). cDNA library size and quality were determined using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Waldbronn, Germany). The libraries were sequenced using an Illumina HiSeq2500 sequencer (Illumina, CA, USA).
Differentially expressed gene (DEG) analysis
Low-quality reads (reads containing more than 10% skipped bases, reads containing more than 40% of bases with quality scores <20, and reads with an average quality score < 20) were filtered from the RNA-seq data using in-house scripts. Filtered reads were identified using the aligner TopHat [24]. Expression levels were calculated using Cufflinks v2.1.1 [24] and Ensembl, release 77. Multi-read-correction and fragbias- correct were used for increasing the measurement accuracy. DEGs were singled out using Cuffdiff with default parameter settings, based on p < 0.05.
Gene Ontology (GO) and pathway analyses
The GO database classifies genes into functional categories and can be used to predict gene function based on Mouse Genome Informatics (MGI) data [25]. Biological changes, related with canonical pathways and functions, were analyzed using an ingenuity pathway (IPA) web-based software (Qiagen, CA, USA). A 1.5-fold change in gene expression and a 1.2-fold change in amino acid levels were used as cut-offs.
Quantitative reverse transcription PCR (RT-qPCR)
To quantify gene expression levels of MNPs@SiO2(RITC) BV2 cells, RNA extraction and cDNA construction for RT-qPCR were performed as previously study [8]. The cells were lysed using RNAzol B (Tel-Test, TX, USA). Chloroform (Sigma-Aldrich, MO, USA) and isopropyl alcohol (Sigma-Aldrich, MO, USA) were used for total RNA precipitation. Pellets were washed with 70% ethanol and the samples were dissolved in RNase-free water. The purities of samples were measured by 260 nm/230 nm and 260 nm/280 nm absorbance ratio using spectrophotometry (Eppendorf, Hamburg, Germany). cDNA was synthesized using the iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The thermal cycle was as follows: 46°C for 20 min followed by 95°C for 1 min.
Expression levels of genes were quantified by qPCR using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) and gene-specific primers (Supplementary Tables 1 and 2) on a Rotor Gene-Q system (Qiagen, CA, USA). Thermal cycles were as follows: 95°C for 5 min, followed by 50 cycles of 95°C for 5 s and 60°C for 30 s. Target gene expressions were calculated with melting curves by the 2−ΔΔCt method.
Gas chromatography (GC)-MS
The amino acid content in the MNPs@SiO2(RITC)-treated BV2 cells and media was analyzed using GC-MS as reported previously [26]. Norvaline is used as internal standard. GC-MS was conducted in both the scan and selected-ion monitoring (SIM) modes on a 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) interfaced with a 5975B mass-selective detector (70 eV, electron impact ionization mode; Agilent Technologies).
Determination of d-serine secreted from microglia
d-Serine concentrations in the medium were determined using a d-serine colorimetric assay (Cosmo Bio, Tokyo, Japan) per the manufacturer’s instructions. Briefly, medium of the MNPs@SiO2(RITC) or lipopolysaccharide (LPS)-treated BV2 cells and primary rat microglia were mixed with NADH and d-serine dehydratase from Saccharomyces cerevisiae (DsdSC), d-serine to pyruvate converting enzyme, at 37°C for 45 min. d-serine samples incubated with nicotinamide adenine dinucleotide (NADH), but without DsdSC were used as blank. The absorbance at 340 nm was measured using a microplate reader (Molecular Devices, CA, USA). Lactate dehydrogenase (LDH) was added to the mixed samples and incubated at 37°C for 45 min. The pyruvate to lactate conversion by LDH leaded to reduction in optical density at 340 nm by NADH oxidation. The d-serine concentration was quantified by calculating the reduction absorbance at 340 nm and a d-serine standard curve.
Liquid chromatography (LC)-MS
l- and d-Serine were purchased from Tokyo Chemical Industry (Tokyo, Japan). HPLC-grade acetonitrile, ethanol, and trifluoroacetic acid were obtained from Thermo Fisher Scientific (CA, USA). Stock solutions of l- and d-serine were prepared at 1 mg/mL in water. A standard stock solution of amino acids was diluted to 0.01–100 μg/mL for calibration. Calibration curves were plotted with seven points using the peak areas obtained for d- and l-serine versus the concentration by linear regression. The samples were centrifuged at 12,000 g for 15 min. The supernatant was filtered through a 3-kDa filter (Millipore, Darmstadt, Germany) by centrifugation at 14,000 g at 4°C for 30 min. The filtered aliquot was used for analysis.
Chiral dl-serine separations were performed using an Agilent 1290 infinitely LC (Agilent Technologies, Walbronn, Germany) using a CROWNPAK CR-I(+) (3.0 × 150 mm, 5 μm, Chiral Technologies) at 30°C. Mobile phase comprising acetonitrile: ethanol: water: trifluoroacetic acid (80: 15: 5: 0.5) was employed at a flow rate of 0.4 ml/min. Two microliters of sample were injected into the column using a thermostated HiP-ALS autosampler. The HPLC system was interfaced with an Agilent 6495 Q-TOF (Agilent Technologies, Walbronn, Germany) mass spectrometer. The source conditions were set to a Vcap of 3.5 kV in positive ESI mode with a sheath gas temperature of 200°C and a sheath gas flow rate of 11 ml/min. The dl-serine was detected by multiple reaction monitoring. A product ion at m/z 60 fragmented by a collision energy of 10V from the precursor ion at m/z 106 was monitored for 200 ms.
Evaluation of neuronal cell viability in a microglia coculture system
The coculture system was set up as reported previously [27]. Briefly, neuronal cells or SH-SY5Y cells were cultured on the bottom side of a Costar Transwell plate (0.4-μm pore size; Corning, NY, USA) in a humidified 5% CO2 incubator at 37°C for 24 h. Microglia in the culture inserts were treated with MNPs@SiO2(RITC) or LPS for 12 h. Then, the inserts were placed in the plate and incubated for another 12 h. To measure the viability of the neuronal cells, the upper inserts were removed, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium solution (CellTiter 96® AQueous One; Promega, WI, USA) was added to each well containing the microglia. The assay plate was incubated at 37°C for 1 h. The amount of soluble formazan produced via cellular reduction was measured using a plate reader (Molecular Devices, CA, USA) at 490 nm. Values were normalized to the corresponding total protein. Images of cell morphology and density were acquired before the cell viability assay under an Axiovert 200 M fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). The excitation wavelength for the MNPs@SiO2(RITC) was 530 nm.
Measurement of the ATP concentration
The ATP concentration of the microglia cocultured neurons were determined using an ATP quantification kit (Promega, WI, USA) per the manufacturer’s protocol. Briefly, the neuronal cells were detached from plate and washed with PBS. Intracellular ATP was extracted with a 1.0% trichloroacetic acid (Sigma Aldrich, MO, USA) at RT for 30 min. The extractant were mixed with a luciferin reagent/buffer mixture and then split in white 384-well plates. After 10 min of incubation at 25°C, the luminescence in each well was measured using a luminometer (LMaxII384; Molecular Devices, CA, USA).
Proteasome activity assay
Proteasome activity was measured using a Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay (Promega, WI, USA) per the manufacturer’s protocol. Briefly, cocultured microglia were washed three times with PBS, replenished with 50 µl of serum-free medium, and left at RT. Proteasome-Glo™ cell-based buffer was mixed with luciferin detection agent and appropriate Proteasome-Glo™ substrate and incubated at RT for 30 min. Fifty-microliter aliquots of this mixture were then added to the medium in each well and incubated at RT for 10 to 15 min. Next, 90 µl of the medium containing a proteasome assay solution was transferred to corresponding wells in a white plate. Proteasome activity was determined by measuring the chymotrypsin-like activity of cellular proteasomes using the luminogenic substrate Suc-Leu-Leu-Val-Tyr-aminoluciferin in a luminometer (LMaxII384; Molecular Devices, CA, USA) per the manufacturer’s instructions.
Evaluation of d-serine distribution and inclusion body formation in vivo
All animal experimental protocols were approved by the Laboratory Animal Research Center of Ajou University Medical Center (approval no. 2020-0033) and complied with the institutional ethical use protocols (NIH Guide for Care and Use of Laboratory Animals). Male ICR mice (8 weeks old, Orientbio, Seongnam, Korea) were maintained under 12-h light/dark cycles with free access to food and water. Four mice per group were used in this study. The biodistribution and toxicological effects of the MNPs@SiO2(RITC) at 25, 50, and 100 mg/kg were previously reported [5]. The study showed the broad-range tissue distribution of the MNPs@SiO2(RITC) and the brain localization of the MNPs@SiO2(RITC) without blood–brain barrier disruption. The particles did not induce significant toxicological symptoms in terms of growth, behaviors, biochemical changes in serum, and histopathology, even at 100 mg/kg. However, based on our in vitro findings, we postulated that there would be subtle toxicity in the brain at 100 mg/kg, which was the maximum concentration evaluated in the previous study. Thus, MNPs@SiO2(RITC) were injected intraperitoneally with sterile saline at 100 mg/kg per mouse. Control mice were injected with sterile saline only. The endpoint was determined at 5 days, based on a previous biodistribution study [5]. Five days after the injection, the mice were anaesthetized with urethane (1.2–1.5 g/kg, intraperitoneally). The hearts were rapidly exposed, and the mice were transcardially perfused with paraformaldehyde (PFA, Sigma-Aldrich, MO, USA). The brains of the PFA-perfused mice were removed and placed in PFA for 24 h. The brains were cryoprotected by immersion in 30% sucrose and then sectioned at 5 μm and stored at –80 °C until analysis.
Frozen sections were incubated in blocking solution containing 1% bovine serum albumin (Sigma-Aldrich, MO, USA) and 10% donkey serum (Sigma-Aldrich, MO, USA) in PBS at RT for 2 h. The sections were stained with anti-d-serine rabbit polyclonal antibody (1:200, Abcam, Cambridge, UK), anti-Iba1 goat polyclonal antibody (1:100, Novus Biologicals, CO, USA), and anti-ubiquitin rabbit polyclonal antibody (1:200, Santa Cruz, CA, USA) diluted in 1% donkey serum in PBS with 0.4% Triton X-100 at 4°C overnight. The sections were rinsed and washed (three times for 10 min) in PBS with 0.4% Triton X-100 and incubated with Alexa Fluor 488- or 647-conjugated secondary donkey antibodies (Invitrogen, CA, USA) at 1:100 dilution in 1% donkey serum in PBS with 0.4% Triton X-100 at RT for 2 h. After the wash steps, the sections were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, CA, USA), and coverslips were applied. Immunostained sections were scanned using a slide scanner (Axio Scan Z1, Carl Zeiss Microscopy GmbH, Jena, Germany), and regions of interest were viewed under a 40× objective lens on an A1R HD25 confocal microscope (Nikon, Tokyo, Japan) at the 3D immune system imaging core facility of Ajou University. Three-dimensional reconstructions of branch structures were acquired and quantified for number and length, using Imaris 9.2 software (Bitplane, Zurich, Switzerland).
Statistical analysis
Data were analyzed by analysis of variance (ANOVA) with Bonferroni’s multiple-comparison tests, using IBM-SPSS software (IBM, NY, USA). Differences were considered significant at p < 0.05.