Main

Nitric oxide (NO) is a neurotransmitter and neuromodulator required for learning and memory.1 NO is generated by NO synthases, a group of enzymes that produce NO from L-arginine. In addition to its normal role in physiology, NO is implicated in pathophysiology. When overproduced, NO combines with superoxide anions (O2·−), byproducts of aerobic metabolism and mitochondrial oxidative phosphorylation, to form peroxynitrite anions (ONOO−) that are highly reactive and neurotoxic. Accumulation of these reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as oxidative and nitrosative stress, respectively, is a common feature of aging, neurodegeneration and Alzheimer’s disease (AD).1

Nitrosative stress caused by peroxynitrite has a critical role in the etiology and pathogenesis of AD.2, 3, 4, 5, 6, 7 Peroxynitrite is implicated in the formation of the two hallmarks of AD, Aβ aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein.1, 4, 7 In addition, peroxynitrite promotes the nitrotyrosination of presenilin 1, the catalytic subunit of the γ-secretase complex, which shifts production of Aβ to amyloid beta (Aβ)42 and increases the Aβ42/Aβ40 ratio, ultimately resulting in an increased propensity for aggregation and neurotoxicity.5 Furthermore, nitration of Aβ tyrosine 10 enhances its aggregation.6 Peroxynitrite can also modify enzymes, such as triosephosphate isomerase,4 and activate kinases, including Jun amino-terminal kinase and p38 mitogen-activated protein kinase, which enhance neuronal cell death.Immunocytochemistry for 3-NT

Neurons were grown on poly-L-lysine coated glass coverslips as previously described38 and fixed with 4% formaldehyde (Ted Pella, Inc.) in PBS for 10 min at RT. Fixed neurons were then permeabilized with 0.1% Triton X-100 in PBS for 5 min. Nonspecific binding was blocked with 3% BSA and 3% FBS in PBS for 1 h at RT. Fixed neurons were then probed with antibodies for 3-NT (1 : 500, Sigma) and an antibody specific for MAP 2 protein (1 : 200, Invitrogen; RT, 2 h), a neuronal marker, followed by conjugated fluorescent secondary antibodies, AlexaFluor594 or AlexaFluor488, respectively, at dilutions of 1 : 500 (RT, 2 h). Chromatin was stained by incubating fixed samples with Hoechst 33342 (1 μg/ml) in PBS at RT for 5 min. To visualize 3-NT using AlexaFluor594, the excitation filter was S555/28 × (Chroma, Bellows Falls, VT, USA) and the emission filter was S617/73m (Chroma). To visualize neurons using AlexaFluor488, the excitation filter was S490/20 × and the emission filter was S528/38m (Chroma). To visualize Hoechst 33342, the excitation filter was S403/12 × and the emission filter was S475/50m. Immunostaining conditions for 3-NT were first optimized along with a blocking control using 10 mM nitrotyrosine to confirm specificity of 3-NT signal. Fluorescence microscopy was performed as previously described.11 Quantification of fluorescence from 3-NT was as follows. Exposure time, brightness and contrast of randomly selected cortical neurons were held constant for all images within same experiment. Using MetaMorph 7.5, a region of interest was selected around each neuron using the MAP 2 label as a guide. This region was transferred to the 3-NT image channel. The fluorescence intensity for each neuron was measured using Show Region Statistics function. Area and intensity/fluorescence data were logged for each neuron. Neurons (25–50) from each treatment were evaluated for a total of over 100 neurons per experiment. Three areas were selected randomly within each image, and the average of their fluorescence intensity was considered as background. The background was subtracted within each image. 3-NT immunofluorescence quantification is expressed as fluorescence per μm2.

APF live-cell imaging

Neurons were cultured on poly-L-lysine-coated MatTek dishes and pretreated with nanoceria for 3 h. To visualize peroxynitrite in neurons, cell permeable APF (2.4 μM; Life Technologies, Carlsbad, CA, USA) was loaded in Neurobasal medium (phenol red-free) containing 0.2% pluronic acid, 1.8 mM CaCl2, 0.8 mM MgCl2 and Hoechst 33342 (1 μg/ml) for 30 min at 37 °C in a humidified 5% CO2 environment. Excess dye was then removed and replaced with conditioned phenol red-free Neurobasal medium. The APF fluorescent signals were measured in response to SNOC (100 μM) at 2 h. Z-stacks were acquired kee** the exposure time, brightness and contrast constant using excitation S490/20 × and emission S528/38m filters (Chroma). Using MetaMorph 7.5 software (Molecular Devices, Sunnyvale, CA, USA), equal backgrounds were subtracted from each z-stack image (as determined from each experiment’s control images), then z-stack series were summed. Cell soma and processes were selected using a region of interest, as previously described.60 This region was transferred to the APF image channel. The fluorescence intensity for each neuron was measured using Show Region Statistics function. Intensity/fluorescence data were logged for each neuron and data were exported to Excel for further analysis.

Nanoceria preparation

Nanoceria were synthesized by a wet chemical process as previously described.61 In brief, to prepare nanoceria with a high ratio of Ce3+/Ce4+, Ce (NO3)3 6H2O (5 mM) was dissolved in dH2O and the nitrate precursor was stirred for 15 min then H2O2 (2% v/v) was rapidly added while stirring at 300 r.p.m. The solution was continuously stirred for 1 h to obtain a stable dispersion of CeO2 nanoparticles. Samples were stored at RT. All preparations were sonicated to ensure single nanoparticles (Branson, Danbury, CT, USA) for 45–60 min before use. For cell experiments, nanoceria were diluted in sterile water.

Transmission electron microscopy

Neurons were cultured on poly-L-lysine-coated 35-mm MatTek glass bottom dishes and fixed with 2% paraformaldehyde, 0.15 M sodium cacodylate, pH 7.4, 2.5% glutaraldehyde for 5 min at RT, followed by an additional 30 min on ice. The fixed cells were then washed three times with ice-cold 0.15 M sodium cacodylate and 3 μM calcium chloride for 3 min on ice, followed by post fixation in 1% osmium tetroxide, 0.8% potassium ferrocyanide and 3 μM calcium chloride in 0.15 M sodium cacodylate for 60 min on ice. After washing cells three times with ice-cold ddH2O for 3 min each, the cultures were stained in 2% uranyl acetate for 30 min on ice. Samples were dehydrated with ice-cold 20, 50, 70 and 90% ethanol and then with 100% ethanol at RT. The samples were first infiltrated in 50% ethanol/50% Durcupan ACM (Fluka/Sigma) for 1 h at RT and under agitation, followed by three changes of 100% Durcupan for 3 h. The resin was polymerized at 80 °C for 3–4 days under vacuum. Sectioning was performed using AO/Reichert Ultramicrotome. Ultrathin (80 nm) sections were post stained with uranyl acetate (5 min) and lead salts (2 min) before imaging using a JEOL 1200FX TEM operated at 80 kV. A subset of sections was imaged without post staining. Negatives were shot at a magnification of × 20 000. The negatives were digitized at 1800 d.p.i. using a Nikon CoolScan system, giving an image size of 4033 × 6010 pixels and a pixel resolution of 0.71 nm.

The nanoparticle morphology was characterized using high-resolution TEM (HRTEM). The nanoceria preparation was deposited on the carbon-coated copper grid (SPI supplies) for HRTEM analysis. The TEM grid was dipped into the nanoceria preparation by the dip-coating technique. HRTEM micrographs were obtained using FEI Tecnai F30 operated at 300 keV.

X-ray photoelectron spectroscopy

Nanoceria were isolated by centrifugation at 20 000 r.p.m using Hermle 220.87 fixed angle rotor in Hermle Z-383K Centrifuge (Cole-Parmer, Vernon Hills, IL, USA) for 20 min and the pellets were dried and resuspended in 10 mM sodium phosphate buffer, pH 7.4, 50 μM DTPA. Samples were then transferred onto silicon wafers (Kmbh Associates, Rancho Cordova, CA, USA; CZ Silicon, thickness of wafer: 350 μm) and air dried. The surface chemistry of the nanoparticles was studied using a Physical Electronics (5400 PHI ESCA) spectrometer with a monochromatic Al Kα X-ray source operated at 300 W and base pressure of 1 × 10−9 Torr. The binding energy of the Au (4f7/2) at 84.0±0.1 eV was used to calibrate the binding energy scale of the spectrometer, and ratios of Ce3+ and Ce4+ in the samples were determined.62, 63

Statistics

Results were collected from three or more independent experiments and are expressed as mean±S.D. Statistical analysis of two populations was compared using two-tailed non-paired Student’s t-test.