Animals

CRISPR-ized mice with TCG deletion in the first exon (Hprt1del8Val) serving as a model of LNS have been previously produced in the Institute of Gene Biology of Russian Academy of Science. In brief, mutation detected in a 9-year-old patient suffering from classical LNS was introduced in CBAxC57Bl6J mice with use of CRISPR/Cas9 system [12]. The experimental groups included 2-month-old mice (determination of uric acid level, studies with acute brain slices and oxygen consumption measurements) and 3-day-old pups (source of primary cultures) all in CBAxC57Bl6J background. The groups included Hprt1del8Val hemizygous males, Hprt1del8Val homozygous females, and WT controls of both sexes. Pups were obtained by breeding of 2-month-old heterozygous Hprt1del8Val females with hemizygous Hprt1del8Val or WT males. Adult mice were maintained in an air-conditioned room (approximately 20–24 °C) in a 12-h light/12-h dark cycle with free access to food and tap water. All the animal procedures were conducted according to the ARRIVE guidelines and approved by the institutional ethical committee of Orel State University (No. 18 dated 21.02.2020) in compliance with Russian Federation legislation.

Determination of Uric Acid Levels

≈200 μL of blood was collected in SiO2-treated tubes via retro-orbital puncture in mice under slight sedation (Zolazepam 5 mg/100 g, i/p). The isolated brains were weighed and frozen at − 80 °C. Tissue samples of 0.5 g were homogenized in 2.5 mL of 0.9% sodium chloride solution in an ultrasonic bath at 30 kHz for 10 min. The tissue was then placed in a homogenizer for 15 min. The homogenate was centrifuged at 14,000 g for 15 min at + 4 °C. Proteins from supernatant were precipitated with acetonitrile or methanol.

To avoid the effect of “false in vitro elevation of the uric acid level” [13], samples were centrifuged (8000 g for 10 min) as soon as possible after collection. The level of uric acid was determined via uricase/peroxidase method with colorimetric detection [14]. The procedure was performed on biochemical analyzer URIT-880 Vet with the use of commercial kits (Olveks Diagnosticum, Cat #012.012) according to the manufacturer’s protocol.

Primary Coculture of Neurons and Glial Cells

Cocultures of cortical and midbrain neurons and glial cells were prepared from pups 3 days postpartum as described [13]. After the decapitation cortex and midbrain were removed into an ice-cold Versene solution (Gibco, UK), the tissue was minced and trypsinized (Gibco, Canada) (0.25% for 15 min at 37°C), triturated and plated on polyethylenimine-coated 22 mm coverslips, and cultured in Neurobasal A medium (Gibco, USA) supplemented with B-27 (Gibco, USA) and 2 mM GlutaMax Supplement (Gibco, USA). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air fed once a week and maintained for a minimum of 12 days before experimental use. Neurons were easily distinguishable from glia: they appeared phase bright, had smooth rounded somata and distinct processes, and laid just above the focal plane of the glial layer. Cells were used at 12–15 days in vitro (DIV) unless otherwise stated.

Acute Brain Slices

Acute brain slices were prepared according to [14]. After the mouse decapitation, the brain was extracted, removed into a cold HBSS (Gibco, USA) (pH 7.4/4 °C) with subsequent performing a sagittal section and preparation of cortical and midbrain slices with a thickness of 300–500 μm. Slices were kept in a cold HBSS with slight oxygenation. Before the experiments, slices were moved into HBSS (pH 7.4/37 °C) for at least 30 min.

Oxygen Consumption Measurements

Intact mitochondria were isolated from brains of WT and Hprt1del8Val mice by a method of differential centrifugation [15] and resuspended in medium containing 250 mM sucrose (Sigma-Aldrich, Germany), 1 mM EDTA (Sigma-Aldrich, USA), and 19 mM Tris–HCl (Sigma-Aldrich, USA) (pH 7.1/25 °C). Oxygen consumption was measured in a Clark-type oxygen electrode (Hansatech, UK) thermostatically maintained at 25 °C containing 135 mM KCl (Sigma-Aldrich, USA), 10 mM NaCl (Sigma-Aldrich, USA), 20 mM HEPES (Gibco, UK), 0.5 mM KH2PO4 (Sigma-Aldrich, USA), 1 mM MgCl2 (Sigma-Aldrich, USA), and 5 mM EGTA (pH 7.1/25 °C). Glutamate (Sigma-Aldrich, USA) (5 mM) and malate (Acros Organics, Belgium) (5 mM) as mitochondrial complex I substrates were added to allow basal respiration (V2). Data were obtained using an Oxygraph Plus system with Chart recording software. Protein levels were determined using Bradford method with bovine serum albumin as a standard [16].

Imaging Mitochondrial Membrane Potential

The \(\Delta \psi m\) was measured by loading of cells with 25 nM tetramethylrhodamine methyl ester (TMRM) (Invitrogen by Thermo Fisher Scientific, USA) in a HEPES-buffered HBSS for 40 min at room temperature. Measurements were obtained by using a Zeiss 900 CLSM (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a × 63 oil immersion objective while keeping 25 nM TMRM in the imaging solution. TMRM was excited using the 561 nm laser line, and fluorescence was measured above 580 nm. Z-stack images were obtained by confocal microscopy, and the basal \(\Delta \psi m\) was measured using Zen Blue software (Zeiss). Assessments of the mitochondrial membrane potential maintenance through application of oligomycin (Sigma-Aldrich, USA), rotenone (Sigma-Aldrich, USA), and the uncoupler FCCP (Sigma-Aldrich, USA) were carried out through recordings from a single focal plane [17]. TMRM was used in the redistribution mode to assess the \(\Delta \psi m\), and therefore, a reduction in TMRM fluorescence represents mitochondrial depolarization.

NADH Measurements

NADH autofluorescence in acute brain slices was measured using the setup with a BDLSMN-375 laser source (Becker & Hickl). The excitation radiation through the optical fiber passes through the collimator and then through the beam splitting filter plate, and the lens is directed to the studied area. Emitted fluorescence light was reflected through a 416 nm long-pass filter to a highly sensitive DCC 3260C cooling CCD camera (Thorlabs, USA) [15].

ROS Assessments

Dihydroethidium (2 μM) (Invitrogen, USA) was used for measurement of cytosolic ROS production. No preincubation (“loading”) was used to limit the intracellular accumulation of oxidized products, and dihydroethidium was presented in the solution during the experiment. Measurements were obtained by using a Zeiss 900 CLSM with a × 63 oil immersion objective. The fluorescence of oxidized form of dihydroethidium was excited by the 561 nm laser line with detection above 575 nm.

For measurement of mitochondrial ROS production, acute brain slices were preincubated with MitoTracker Red CM-H2Xros (1 μM) (Invitrogen, USA) for 30 min at 37 °C. Fluorescence intensity measurements were produced using 561 nm excitation and emission above 570 nm [18].

Glutathione Assessments

Cocultures of neurons and glial cells were incubated with 50 μM monochlorobimane (MCB) (Invitrogen, USA) for 40 min in HEPES-buffered salt solution prior to imaging [19]. Cells were then washed with HEPES-buffered salt solution, and images of the fluorescence of the MCB-GSH were acquired using a Zeiss 900 CLSM with excitation at 405 nm and emission at 435–485 nm.

Genotyping

Breeders and pups were genotyped with the use of TaqMan system. DNA was extracted from tails via digestion in fresh alkaline lysis buffer (10 mL sterile dWater, 14 μL 50% sodium hydroxide, 14 μL 0.5 M EDTA, pH 8.0) and purification with the use of DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. PCR was performed on the CFX-96 (Bio-Rad, USA) platform. The primers used were as follows: F: 5′-CTTTTTGCCGCGAGCCGACC-3′; R: 5′-GCCTGCGTCACTGTGCCAC-3′. TaqMan probes used were as follows: ROX-5′-CAGTCCCAGCGTGGTGAGCCAA-3′-BHQ1 for mutation and FAM-5′-TCCCAGCGTCGTGGTGAGCCAA-3′-BHQ1 for WT allele.

Statistical Analysis

Data were analyzed with Origin Pro 2018 (MicroCal, Oregon, USA) and are expressed as mean ± SEM. The Mann–Whitney test was used to estimate the statistical significance between experimental groups. Significance was accepted at a 95% confidence level (\(p <0.05\)). *\(p < 0.05\), **\(p < 0.001\), and ***\(p < 0.0001\).