IsoQC (QPCTL) knockout mice suggest differential substrate conversion by glutaminyl cyclase isoenzymes
Supplementary Information
Correspondence to: Stephan Schilling,Fraunhofer Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation (IZI-MWT), Weinbergweg 22, 06120 Halle/Saale, Germany
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Supplementary Methods
Behavioral analysis
The pole was used as a simple test for motor-coordinative deficits. The test item consisted of a metal pole (diameter: 1.5 cm; length: 50 cm) wrapped in anti-slip tape and topped with a plastic ball that was vertically installed on a heavy platform. For testing, the animals were placed head-up directly under the ball. Time to realign and descend the pole was measured (cut-off time: 120 s) with aberrant activities (e.g. falling, jumping, sliding) recorded as 120 s. The best performance over five trials was used for analysis. In addition, the rotarod test was used to investigate neuro-motor performance. It provides a quantitative assessment of coordination and balance, since animals must continuously walk on a horizontal, rotating cylinder to avoid falling off the rod. Testing was performed on two consecutive days, using a computer controlled RotaRod System (TSE Systems, Bad Homburg, Germany). In the first morning session mice were trained on a constantly rotating rod (10 revolutions per minute (rpm)) until they were able to stay on the drum for at least 60 s. In the afternoon and on the following day, three test sessions were conducted consisting of three trials each. The rod-speed was accelerated from four to 40 rpm over a five-minute period. The system automatically recorded the total distance covered by the animal before it fell off the rod. Performance was examined by analysis of each trial (motor learning), and by best trial analysis (motor coordination).
Automated phenotyping
An eight-place TSE Phenomaster System (TSE Systems, Bad Homburg, Germany) was used to assess the circadian pattern of locomotor activity and ingestion behavior as well as indirect calorimetric parameters in a home cage environment.
Spontaneous ambulatory movements (XY plane) and rearing events (Z plane) were detected by infrared light beam frames surrounding each cage (IVC Green Line cages, Tecniplast, Italy). Two dedicated weighing sensors per cage registered ad libitum water and food consumption. Continuous mode calorimetry via one O2/CO2 gas sensor pair per cage allowed the simultaneous calculation of O2 consumption, CO2 production, respiratory exchange rate (RER = VCO2 [ml/h/kgBW] / VO2 [ml/h/kgBW]) and heat (performed by TSE PhenoMaster Software, version 4.8.9.). All gas sensor pairs were calibrated with calibration gas mixtures (CO2, 0.05%, O2 20.895%, in N2; CO2 0.950%, O2 20.00%, in N2) before each test session.
Mice were individually observed for 3 days under a 12-h light/dark cycle (lights on, 5:30 A.M.; lights out, 5:30 P.M.). Two sets of animals were tested at the age of 47 weeks: 8 males (4 isoQC+/+, 4 isoQC-/-) and 8 females (4 isoQC+/+, 4 isoQC-/-). Data were collected automatically with a rate of 100 Hz and stored as a sum over 1-min intervals.
Results were pooled and analyzed using two-way repeated-measures ANOVA, with the factor genotype representing the inter-individual factor and time representing the intra-individual factor.
Analysis of splenocytes
Mice were transcardially perfused using saline. Subsequently, the spleen was removed and pored through a filter of 40 µm pore size. The cells were suspended in a staining buffer consisting of phosphate-buffered saline, 0.5% (w/v) bovine serum albumin, 2 mM EDTA and subjected to centrifugation (300 x g, 15 min). The pellet was resuspended in 420 µl of buffer and labeled MicroBeads (Miltenyi Biotech) were added (T-lymphocytes: CD 90.2, B-lymphocytes: CD45R/B220 MicroBeads) for later separation and detection of the cells. The differentially labeled cells were separated (magnetic field) and fractionated in different tubes and subsequently subjected to antibody staining. After incubation at 4°C for 15 min, the suspension was washed using 10 ml of staining buffer for later fluorescence-activated cell sorting (FACS) analysis. Supplementary Table 1 below provides an overview about the antibodies applied (all from Miltenyi biotech). All samples were analyzed using a FACSCalibur (BD Biosciences, CA).
Supplementary Table 1. Antigens and fluorescence tags of antibodies applied for splenocyte analysis by FACS. Abbreviations: FITC, fluorescein-isothiocyanate; PE, phycoerythrine; APC, allo-phycocyanin
Fluorescence label/tube / FITC / PE / APCTube 1 / CD3e / CD49d / CD45R/B220
Tube 2 / 7/4 / Ly6G / CD11b
Tube 3 / CD4 / CD25 / CD8a
Tube 4 / CD3e / NKp46
Supplementary Figure 1. Phenotypic characterization of mice of isoQC-/- mice. a-e, Results of intrahome-cage-automated phenotyping (n = 8 per group, 65h; DP, dark period; LP, light period): (a, b) Food and water consumption, normalized to body weight. (c) Activity of isoQC mice in home cage environment. (d) Average CO2 production. (e) Respiratory exchange ratio (RER). Statistically significant differences have not been observed in any of the parameters. (f) Rotarod analyzing the motor performance of the mice. Mice of the isoQC ko line were indistinguishable from wt littermates with regard to motor performance (n ≥ 5 per genotype).
Supplementary Figure 2. Analysis of splenocytes from isoQC+/+ and isoQC-/- mice. Spleen is a tissue with significant isoQC activity (compare with Figure 1). In order to analyze, whether the depletion in isoQC might affect the composition of different immune cells in spleen, splenocytes were isolated and characterized by FACS analysis after fractionation. Significant differences between isoQC+/+ and isoQC-/- mice were not observed. Thus, the depletion of isoQC does apparently not affect the development of different immune cell populations.