Mouse strains
Homozygous Pink1−/− plus SNCA A53T double mutant mice were generated by crossing Pink1−/− mice (background: 129/SvEv) with A53T-SNCA-overexpressing PrPmtA mice (background: FVB/N) and then, interbreeding the littermates. They contain 129/SvEv and FVB/N genetic backgrounds approximately in a 50:50 distribution. Wildtype (WT) control mice are hybrids from a crossbreeding of 129/SvEv and FVB/N mice, which were descended from littermates of the respective single mutant animals. The double mutant mice are referred to as Pink1−/−SNCAA53T or in short, Pink1SNCA.
Mice had free access to food and water, and they were maintained in climate-controlled rooms with a 12 h light-dark cycle. Behavioral experiments were performed between 10 am and 3 pm. The experiments were approved by the local Ethics Committee for animal research (Darmstadt, Germany), adhered to the guidelines for pain research in conscious animals of the International Association for the Study of PAIN (IASP) and those of the Society of Laboratory Animals (GV-SOLAS) and were in line with the European and German regulations for animal research.
Analysis of nociception and somatosensory functions
Nociceptive tests were performed at 2, 4, 10 and 12 months of age with 8 mice per group. Mice were habituated to the test room and the test chambers for three consecutive days before the baseline measurement.
The latency of paw withdrawal on pointy mechanical stimulation was assessed using a Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio, Italy). The steel rod was pushed against the plantar paw with ascending force (0–5 g, over 10 s, 0.2 g/s) and then maintained at 5 g until the paw was withdrawn. The paw withdrawal latency was the mean of three consecutive trials with at least 30 s intervals.
The sensitivity to painful heat stimuli was assessed by recording the paw withdrawal latency with a Hot Plate (52 °C or 30–55 °C surface, Föhr Medical Instruments, Germany) or with the Hargreaves test (IITC Life Science). In the latter, an infrared lamp was placed with a mirror system underneath the respective hind paw. By pressing the start button the lamp starts to emit a heat-beam until the paw is withdrawn, which stops the lamp. The mean paw withdrawal latency of three tests with at least 10 min intervals was used for statistical analysis.
The Orofacial Pain Assessment Device (OPAD, Stoelting, Ireland) allows for evaluation of facial thermal nociception by using a reward-conflict paradigm. The OPAD cage consists of a plexiglas chamber with metal grid floor and an adjustable slit giving access to the nipple of the reward bottle, which is flanked with two PC-controlled thermal peltier elements. To receive the reward (diluted milk in water), the mouse has to touch the thermodes with its cheeks. Hence, the mouse has to decide between receiving a reward or escaping the aversive heat or cold stimulus. Mice were fasted overnight to increase the appetite. Mice were trained three times a week for two weeks at innocuous temperatures (36.5–38 °C) to get a stable baseline of at least 600 licks in 18 min. During test periods, temperature circles starting from neutral (37 °C, 3 min) to aversive cold (15 °C, 10 °C each 3 min) or aversive heat (45 °C, 54 °C each 3 min) were applied using a ramping protocol. Circles were repeated up to 20 min. The ANY-maze software (Version 4.99, Stoelting) registered licks and contacts with the thermodes. The average and total numbers of lickings and contacts at the defined temperatures were used for analysis. Ramping times were excluded.
Analysis of olfactory functions
The Novel Odor Recognition Test (NOdorR) was used to assess whether mice were able to detect and differentiate between odors. Mice were habituated to a 3-chambered test box (sizes as for social tests) for 3 days, each for 10 min. The test sessions consisted of three different stages. During (1) habituation, mice were again adapted to the box for 10 min. During (2) "Learning", two petri dishes with perforated lids containing 2 × 2 cm filter papers soaked with identical odors (1% vanilla) were placed in both side chambers. The mouse was placed in the middle and was allowed to explore odors for 10 min. In (3) "Novelty", the settings were identical to the learning stage except that one of the familiar odors (1% vanilla) was replaced with a novel one, which was undiluted rose water. After each test session, the open field was carefully cleaned. Filter papers and petri dishes were freshly prepared for each mouse. Visits of chambers, visits of odors and the time spent with odors were recorded with the VideoMod2 Software. In analogy, odor preferences were tested for urine mixtures of stranger mice.
Analysis of social cognition and memory
The social test apparatus consisted of a rectangular, three-chambered box (42.5 cm wide, 22.2 cm high) with a central chamber 17.8 cm in length and two side chambers each 19.1 cm in length, according to recommended specifications [78]. The central chamber was separated from the side chambers by removable partitions with doors allowing the animal to move freely between chambers. The "stranger" stimulus mouse was positioned in a grid enclosure (Ugo Basile, Italy) allowing close interactions and nose contact but preventing the stranger mouse from initiating social contact. The enclosures had an internal diameter of 7 cm, and height of 15 cm. The top and bottom were constructed from grey PVC.
We used two stimulus mice in each experiment with the same gender and strain as the test mouse, but housed in another room. Mice were habituated to the test box for 3 days, each for 10 min. The sessions were similar as for the NOdorR: (1) Habituation: the animal was placed in the middle chamber with the dividers closed to allow exploration of the middle chamber for 5 min. (2) Sociability test: after the 5-min habituation period, an unfamiliar adult gender-matched mouse (stranger 1) was placed inside the grid enclosure in one of the side chambers. An identical empty grid enclosure was placed in the opposite chamber. The dividers were then raised, allowing the test mouse to move freely among the chambers over a 10-min test session. (3) Novelty: The original stranger mouse (stranger 1) remained in its grid enclosure on one side of the apparatus. A new unfamiliar mouse (stranger 2) was placed in the grid enclosure on the opposite side. The behavior was observed for 10 min. Visits of strangers and the time spent with strangers were recorded. A "visit of stranger" required close proximity of the nose with the grid enclosure with a maximum distance of one cm.
Phenomaster
The TSE Phenomaster offers an automated metabolic and behavioral monitoring in home cage environments. Drinking and feeding behavior were monitored with high-precision weight sensors for liquid and food dispensers, which are integrated into the lid of the cage. Mice were adapted to the drinking bottles for one week in their home cage and to the Phenomaster® cage for 2 consecutive days before starting the experiment. Drinking, feeding and voluntary wheel running were recorded for 24 hours.
Analysis of motor functions
Motor coordination and endurance were assessed with the accelerating RotaRod test (16–32 rpm, ramp 3 rpm/min, cut-off 5 min; Ugo Basile, Italy) or a RotaRod at constant speed. Mice were trained with 2–3 training runs. The running time in test trials was averaged from three trials. The cut-off time was 300 s.
Motor functions of the front limbs were assessed with the mouse staircase test (Campden Instruments Ltd., UK). The test apparatus comprises two compartments, a start compartment with a hinged lid and a test compartment containing a small central platform with two staircases (8 steps) on each side. Each step has a cup for a reward, which was filled with sweet pellets. At the beginning, mice were set on a restriction diet to increase the appetite. Mice were adapted to the sweet rewards for three days in the home cage (d 1–3) and in two training trials (d 5–6). Test sessions were performed at day 7 to 9 and lasted 30 min. Mice were placed into the test compartment, and the behavior to grasp, lift and collect food was monitored with a video camera, and analyzed with VideoMod2. The numbers of pellets remaining on the steps or floor were counted.
Grip strength was assessed with a computerized Grip-Strength Meter (Model 47200, Ugo Basile, Italy). The apparatus consists of a grasping grid connected to a force sensor. To measure the grip strength of front limbs and/or hind limbs, mice were held by the tail and allowed to grasp the grid. As soon as the mouse grasped the grid, the mouse was pulled backwards by the tail until the grip was lost. The peak force of each measurement was automatically recorded in gram-force (gf) by the software. The grip strength was the mean of three trials.
Culture and staining of primary DRG neurons
Primary adult dissociated DRG neuron-enriched cultures were prepared by dissecting mouse dorsal root ganglia (DRGs) into 1x PBS (Phosphate Buffered Saline, Gibco, Germany), followed by digestion with 5 mg/ml collagenase A and 1 mg/ml dispase II (Roche Diagnostics, Germany). Triturated cells were centrifuged through a 15% BSA (bovine serum albumin) solution and plated on poly-L-lysine and laminin coated cover slips in Neurobasal medium (Gibco) containing 2% (vol/vol) B27 supplement (Gibco), 50 µg/ml Pen-Strep, 100 ng/ml NGF and 200 mM L-glutamine. After incubation for 2 h, 2 ml Neurobasal medium was added and neurons were cultured for up to 48 h depending on the experimental requirements. Cells were kept at 37 °C, 5% CO2, 95% humidity. Primary DRG neurons were used for calcium imaging and immunohistochemistry stainings.
Calcium Imaging
Calcium fluxes were measured fluorometrically as the ratio of the absorbances at 340 and 380 nm (F 340/380) in cultured adult DRG neurons (see DRG primary cultures). Calcium-imaging experiments were performed with a Leica calcium-imaging setup, consisting of a Leica DMI 4000 b inverted microscope equipped with a DFC360 FX (CCD) camera, Fura-2 filters, and an N-Plan 10x/0.25 Ph1 objective lens (all from Leica). Images were captured every two seconds and were processed with the LAS AF-software (Leica). Cells were loaded with 5 µM of the Ca2+-sensitive fluorescent dye Fura-2-AM-ester (Biotium), incubated for 40 min at 37 °C and washed three times with ringer solution (Fresenius). Coverslips were then transferred to a perfusion chamber with a flow rate of 1–2 ml/min at room temperature. Baseline ratios were recorded with ringer solution for 100–180 s, before application of either 0.01% formalin to activate TRPA1 or 50 nM capsaicin (Sigma) to activate TRPV1 ion channels for 100 s or 26 s, respectively. After wash-out with ringer solution, cells were perfused with 100 mM KCl (high K+) to assess depolarization-evoked calcium currents and the viability of the neurons. Data are presented as changes in fluorescence ratios (F340/380) normalized to baseline ratios. The analysis encompassed 300–350 neurons per condition of 10–12 independent DRG cultures of each three mice per group per stimulus, which were 18 months old. The maximum, the time of maximum and area of the fold increase versus time curve was calculated by integration (Origin Pro 2020 software) with the baseline fixed at Y = 1. The time courses and areas were used for statistical comparison.
Quantitative real-time PCR (QRT-PCR)
Mice were sacrificed via carbon dioxide followed by rapid blood withdrawal. The dorsal root ganglia were rapidly dissected and frozen in liquid nitrogen. Total RNA was extracted from mouse tissue and reversely transcribed with random primers. Twenty nanograms of cDNA equivalent were subjected to quantitative real-time polymerase chain reaction (qRT-PCR). The PCR reaction and amplicon detection were done on a TaqMan (AB 7500 Applied Biosystems; Life Technologies Corporation, Carlsbad, CA) using a FastStart Universal Master Mix (Roche Diagnostics, Mannheim, Germany) with SYBR Green fluorescence staining. TRP channel mRNA were amplified with specific primers and were normalized to the housekeeping gene protein phosphatase 1 (PPP1CA). The comparative threshold cycle (CT) method was used for quantification of relative mRNA expression. The primer pairs are summarized in Suppl. Table 1a.
Immunohistochemistry
Mice were terminally anesthetized with isoflurane and cardially perfused with cold 0.9% NaCl followed by 2% paraformaldehyde (PFA) for fixation. Tissues were excised, postfixed in 2% PFA for 2 h, cryoprotected overnight in 20% sucrose at 4 °C, embedded in small tissue molds in cryo-medium and cut on a cryotome (10 µm for DRGs and ScN, 12 µm for SC). Slides were air-dried and stored at − 80 °C. After thawing, slides were immersed and permeabilized in 1xPBS with 0.1% Triton-X-100 (PBST), then blocked with 3% BSA/PBST, subsequently incubated overnight with the first primary antibodies in 1% BSA/PBST at 4 °C. After washing three times with PBS, slides were incubated with the secondary antibodies for 2 h at room temperature, followed by 10 min incubation with DAPI and embedding in Aqua-Poly/Mount. The general settings were optimized for the respective antibodies and tissues. Primary antibodies included STING, IFI16, Tuji, Ceramides, SNCA (Suppl. Table 1b). Secondary antibodies were labeled with fluorochromes (Invitrogen, Sigma, Life Technologies). Slides were analyzed on an inverted fluorescence microscope (BZ-9000, KEYENCE, Germany and Axio Imager Z1, Zeiss, Germany). Tiled images were obtained to reconstruct whole DRG or spinal cord sections to assess different regions of DRG neurons and fibers.
For analysis of neurite outgrowth and morphology, primary neuron cultures were washed in PBS, fixed in 4% PFA and immunostained with antibodies directed again neurofilament of 200 kDa and subsequent Alexa-488 or Cy3 labeled secondary antibodies, and with phalloidin-Alexa-594. Images were captured on an inverted Axio Imager Z1 fluorescence microscope (Zeiss, Jena, Germany). Tiled images were obtained to reconstruct the whole cultures and assess density of neurons and dendrites.
For quantification, RGB images were converted to binary images using threshold setting implemented in ImageJ with minor adjustments. The particle counter of FIJI ImageJ was used to assess the area covered with neuronal immunoreactive structures.
Transmission electron microscopy
Mice were terminally anaesthetized with carbon dioxide and perfused transcardially with cold 0.9% sodium chloride (NaCl) followed by perfusion with the fixation solution containing 4% PFA and 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. DRGs were excised and postfixed in the fixation solution for 90 min. After washing 3 times with 0.1 M cacodylate buffer, the tissue was osmicated (1% OsO4 in 0.1 M cacodylate buffer) for 60 min, washed 3 times with 0.1 M cacodylate buffer and dehydrated in ethanol, 1% uranyl acetate and propylene oxide. The tissue was then incubated in Durcupan resin (Fluka, Durcupan ACM-Kit) overnight and embedded in fresh Durcupan resin at 56 °C for 48 h. Ultrathin sections (54 nm) were cut with a diamond blade, collected on single slot Formvar-coated copper grids and contrasted with lead citrate. Sections were analyzed using a Zeiss electron microscope (Zeiss EM900) and imaged with a slow-scan CCD-Camera.
Oxygraph analysis of mitochondrial OXPHOS activity
Brain sections (prefrontal cortex, hippocampus) and DRG plus trigeminal nerve plus ganglion were immediately transferred into ice-cold Respiration medium MiR06Cr containing 280 U/ml catalase (Pesta&Gnainer, 2012). Tissue was homogenized using a motor-driven tightly fitting glass/Teflon Potter-Elvehjem homogenizer. Mitochondrial respiration was measured using high-resolution respirometry (Oxygraph-2 k, Oroboros Instruments, Innsbruck, Austria) with DatLab software 6.1.0.7 (Oroboros Instruments, Innsbruck, Austria). LEAK-respiration was induced by the addition of complex I –linked substrates pyruvate (5 mM), malate (0.5 mM) and glutamate (10 mM). Complex I – linked respiration was measured after adding ADP (2.5 mM) in a saturating concentration. To measure complex II-linked respiration, rotenone (0.5 µM) was added to block complex I followed by the addition of succinate (10 mM). Maximum uncoupled respiration (ETS, electron transfer system capacity) was measured after stepwise titration of FCCP ((carbonyl cyanide-p-trifluormethoxyphenylhydrazone). Residual oxygen consumption (ROX) was determined after sequential inhibition of complex III with Antimycin A and complex IV with azide. Absolute respiration rates were corrected for ROX and normalized for the protein content.
Lipid analyses
Sphingolipids and ceramides were analyzed in plasma and in tissue of the spinal cord (SC), dorsal root ganglia (DRG) and sciatic nerves (ScN) of aged Pink1SNCA and wildtype control mice. Lipid analyses were done using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), according to procedures described in detail in [79]. The analysis encompassed dehydro-ceramides (Cer d18:0/16:0, Cer d18:0/18:0, Cer d18:0/24:0, Cer d18:0/24:1), ceramides (Cer 18:1/16:0, Cer 18:1/18:0, Cer 18:1/20:0, Cer 18:1/22:0, Cer 18:1/24:0, Cer 18:1/24:1), glucosyl- (GlcCer 16:0) and lactosylceramides (LacCer 18:0, LacCer 24:0, LacCer 24:1), sphinganines (d18:0 SPH, d20:0 SPH), sphingosine (d18:1 SPH) and the respective phospho-SPH's (d18:0 S1P, d18:1 S1P).
In brief, tissue samples were homogenized by adding ethanol:water (1:3, v/v, tissue concentration 0.02 µg/ml) using a ball mill mixer (MM400, Retsch, Haan, Germany) with 4 zirconium oxide grinding balls (25 Hz for 2.5 min). Subsequently, 50 µl of the homogenate were extracted using a liquid-liquid-extraction method. Plasma sample volumes were 10 µl. The quantification of all analytes was performed using a hybrid triple quadrupole-ion trap mass spectrometer QTRAP 5500 (Sciex, Darmstadt, Germany) equipped with a Turbo-V-source operating in positive ESI mode. Sphingolipids were separated using an Agilent 1290 HPLC system equipped with a Zorbax C18 Eclipse Plus UHPLC column (50 Å~ 2.1 mm, 1.8 µm, Agilent technologies, Waldbronn, Germany). Quality control samples of three different concentration levels (low, middle, high) were run as initial and final samples of each run. For all analytes, the concentrations of the calibration standards, quality controls and samples were evaluated by Analyst software 1.6 and MultiQuant Software 3.0 (Sciex) using the internal standard method (isotope-dilution mass spectrometry). Calibration curves were calculated by linear or quadratic regression with 1/x weighting or 1/x2 weighting. Variations in accuracy of the calibration standards were less than 15% over the range of calibration, except for the lower limit of quantification (LLOQ), where a variation in accuracy of 20% was accepted. Lipid concentrations in tissue are expressed as pg/mg of the tissue or ng/ml of plasma. For multivariate statistical analyses, lipid data were normalized on the mean value of the respective tissue, and they are expressed as percentages of the mean because the concentrations of different lipids differ by several orders of magnitude.
Statistics
Group data are presented as mean ± SD or mean ± sem, the latter for behavioral time course data, specified in the respective figure legends. Data were analyzed with SPSS 24 and Graphpad Prism 8.0 and Origin Pro 2020. Data were mostly normally distributed, or log-normally distributed. For testing the null-hypothesis that groups were identical, the means of two groups were compared with 2-sided, unpaired Student's t-tests. The Mann Whitney U test was used as a non-parametric alternative in case of violations of t-test requirements. Time course data or multifactorial data were submitted to 2-way analysis of variance (ANOVA) using e.g. the factors 'time' and 'genotype'. In case of significant differences, groups were mutually compared at individual time points using post hoc t-tests according to Dunnett, i.e. versus the control group, or according to Šidák. In case of violations of sphericity, degrees of freedom were adjusted according to Huynh Feldt. Asterisks in figures show multiplicity-adjusted P-values. The areas under the curve of calcium imaging data were log-normally distributed and were Log2-transformed for statistical comparisons. The areas were calculated by integration and the distribution was obtained by Kernel density estimation (Origin Pro).
Principal component analysis was used to define the lipid species, which accounted most for the variance between genotypes. Further multivariate analyses included canonical discriminant analysis to assess the predictability of group membership and separation of genotypes and tissues. Partial least square analysis was used if analytes exceeded the number of samples per group. The statistical analysis of RNAseq data is explained below.
RNA sequencing and analysis
DRGs were removed and flash-frozen on dry ice. RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (TruSeq Total RNA with Ribo-Zero rRNA depletion) was used with 1 µg of total RNA for the construction of sequencing libraries. Libraries were prepared according to Illumina's instructions. Sequencing was performed with an Illumina Next Generation sequencing system with a sequencing depth of 50 Mio reads per sample.
Sample quality was assessed with demultiplexed fastq.gz files and subsequently the alignment was performed with SeqMan NGen 16 (Lasergene) using the reference genome mm10 provided from UCSC (GRCm38) [80] as template, a minimum read length of 35 bp and automatic adapter trimming. Results were displayed with ArrayStar 16 (Lasergene) including the amount of mapped reads, target length, source length and position, strand, genes and gene IDs, annotated according to the mm10 assembly, and reads were normalized according to RPKM. Alternative DEseq2 normalization yielded equivalent results. Normalized reads were analyzed with ArrayStar, which uses general linear models to assess differential expression. Genes were filtered for at least 10 valid values out of 17 samples with normalized reads > 0.05 to exclude low expression genes. Data were log2 transformed, single missing values were imputed from the normal distribution, and results were displayed as scatter plots, MA-plots and Volcano plots, the latter showing the log2 difference i.e. fold change (positive for upregulated genes and negative for downregulated genes) versus the –log10 of the t-test P value. The P value was set at 0.05 and adjusted according to Benjamini Hochberg. Hierarchical clustering was employed to assess gene expression patterns using Euclidean distance metrics. Results were displayed as heat maps with dendrograms.
Key regulated genes (based on P-value, fold change and abundance) were further analyzed for gene ontology annotation enrichments for 'cellular component', 'biological process' and 'molecular function', KEGG, Biocarta and Reactome pathways, SMART domains and SP-PIR-keywords to assess common localizations and functions of significantly regulated genes. GO analyses were done with the "term enrichment" and "functional gene clustering" tools of The Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8) (http://david.abcc.ncifcrf.gov/home.jsp) [81]. In addition, gene set enrichment analyses (GSEA) (http://www.gsea-msigdb.org) [82] were used to further assess functional implications of up- or downregulated genes and to obtain a gene ranking and heat map of the leading edge 50 up- and downregulated genes. GSEA generates ranked gene lists based on fold difference and P value. The RNAseq data have been deposited as GEO dataset with the provisional accession number GSE146091.