Esse in actu, Esse ut actus y la redimensión de la esencia

2.2.6.1. Sample preparation

Cells grown in tissue culture plates were trypsinized and harvested at 106 × g for 5 min at 4 ºC. Cell pellets were then washed with chilled PBS and dried with 1-propanol under nitrogen flow.

For the extraction of epidermal lipids, skin biopsies from the back of CerS3 deficient mice and control littermates were rapidly dissected and snap-frozen in liquid nitrogen before being stored at – 80 ºC. Skins were thawed and epidermis were isolated by treating the biopsies with 500 µg/ml thermolysin buffer for 2 h at 37 °C (Germain et al., 1993). Afterwards, epidermis was separated from dermis, cut into small pieces, and lyophilized (Alpha 1-2, Christ).

2.2.6.2. Lipid extraction by modified BLIGH and DYER method

Sphingolipids were extracted according to Doering et al. with slight modifications (Doering et al., 1999a; Jennemann et al., 2007). In general, lipids were isolated using mixtures of CHCl3/CH3OH/H2O.

In particular, dried pellets from cultured cells were extracted with 2 ml 10/10/1 (v/v/v) solvent mixture at 37 ºC for 15 min with occasional sonication. After centrifugation at ~ 2000 × g for 10 min, supernatants were collected and pellets were then reextracted once more with 10/10/1 solvent mixture, and finally with a 30/60/8 (v/v/v) solvent mixture.

For the extraction of epidermal free lipids, ~ 3 mg of dried weight epidermis cut in small pieces was extracted once with CHCl3/CH3OH/H2O 30/60/8, then with 10/10/1 and finally with CHCl3/CH3OH 2/1 (v/v/v) as described. Each extraction step was performed at 50 ºC for 15 min under sonication.

Supernatants were combined and dried under a nitrogen flow at 37 ºC. In parallel, pellets were dried and kept at 4 ºC for further analysis.

2.2.6.3. Removal of phospholipids by mild alkaline methanolysis

Combined lipid extracts were subjected to methanolic mild alkaline hydrolysis (0.1 M KOH in CH3OH) for the removal of phospholipids. The saponification of cell culture extracts was performed at 37 ºC for 2 h, while epidermal extracts required 4 h at 50 ºC for complete hydrolysis. Sequentially, saponified lipid extracts were neutralized with glacial AcOH and solvent was removed under a mild nitrogen flow.

2.2.6.4. Desalinization by reverse-phase chromatography (RP-18)

Saponified lipid extracts were desalted by reverse phase chromatography prior to being analyzed either by TLC or ESI-MS/MS. Thus, columns packed with C18 material (Porasil silica 125Å 55–105 µm) were preconditioned consecutively with 3 times CH3OH and 2 times with 0.1 M KCl. Salt-containing samples were dissolved in ddH2O to a final concentration of 0.1–0.2 M KCl by brief sonication before being loaded into the columns. Following the loading of the samples, vials were washed twice with 0.1 M KCl, sonicated and loaded as well into the columns. Lipids bound to the column were then washed 3 times with ddH2O. Finally, sphingolipids were eluted

with CH3OH and dried under a nitrogen flow. In case of desalinization of epidermal ceramides, an additional washing step with ddH2O was performed prior to elute the lipids with CH3OH into the original sample tubes, and subsequently dried under a nitrogen flow.

Desalting of saponified lipids from cultured cells was performed using freshly prepared Pasteur pipettes. For tissue extracts, 100 mg of packing material was loaded into 5 ml polypropylene reusable columns. To assure complete removal of previous lipid extracts, polypropylene columns were carefully washed with 2 x 4 ml CHCl3, 1 x 4 ml CHCl3/CH3OH 1:1, 1 x 4 ml CH3OH, 1 x 4 ml CH3OH /H2O 1:1, and finally equilibrated with 2 ml 0.1 M KCl.

2.2.6.5. Extraction of protein-bound sphingolipids

Protein-bound sphingolipids from epidermal biopsies were extracted from the pellets obtained after extraction of free lipids. Pellets were “washed” 3 times with 2 ml of 100% CH3OH for 5 min and 2 times with 2 ml of 95% CH3OH at 60 °C for 2 h to remove residual free lipids. Treatment of the residual pellets with 1 ml of 1 M KOH in 95% methanol at 60 °C for 2 h cleaved ester linkages and released protein-bound sphingolipids. Supernatants were neutralized with AcOH and desalted using RP-18 columns as described above.

2.2.6.6. Extraction of fatty acids

Fatty acids rich in ULC-acyl moieties were isolated from GM2AP^-/- mice testes by acid hydrolysis, as previously described (Valianpour et al., 2003). Following homogenization and lyophilization of the biopsies, fatty acids were hydrolyzed at 90 ºC for 2 h with 10 ml of CH3CN/HClconc. 4/1 (v/v). Testicular fatty acids were then extracted with the double amount of n-hexane. The upper organic phase was separated and dried under a nitrogen flow. For cell culture experiments, isolated fatty acids corresponding to 2 mg of wet weight were dissolved first in ethanol (1 mg wet weight / 1 µl ethanol), following intensive vortexing and finally dissolved in 2 ml of culture media.

2.2.6.7. Protein determination according to LOWRY

Pellets obtained after lipid extraction were used for determination of their protein content according to the method of Lowry (Lowry et al., 1951).

This colorimetric assay is based on the redox-reaction of peptide bonds with CuSO4 under basic conditions and the subsequent reduction of Folin-Ciocalteu reagent by Cu⁺. First, proteins dissolved in alkaline conditions react with Cu²⁺ forming a tetradentate Cu²⁺ complex with the amino groups of peptide bonds. Subsequently, Cu²⁺ is reduced by certain peptide side chains. This reaction is stabilized by tartrate.

Upon addition of the Folin-Ciocalteu reagent containing a mixture of phosphomolybdate and phosphotungstate (Mo⁶⁺/W⁶⁺), reduction to heteropolymolybdenum blue (Mo⁵⁺ and Mo⁶⁺) takes place, which is detectable at a maximum of 750 nm.

Therefore, pellets were dissolved in 1 M NaOH at 50 ºC for 4 h with occasional sonication. To determine the protein concentration, a standard curve was prepared with BSA (0–20 µg) in 0.1 M NaOH. Sample aliquots and BSA standards were dissolved in 0.1 M NaOH to a final volume of 300 µl. Complex reagent (1.5 ml) containing Na2CO3, CuSO4, and tartrate was added to each sample. Copper complexes were allowed to form by incubating the samples 10 min at room temperature. Subsequently, 150 µl of Folin-Ciocalteu reagent were added, followed by a brief vortexing. Protein complexes were then incubated 45 min at r.t. in the dark, and protein content was measured by spectrophotometry at 750 nm.

2.2.6.8. Quantification by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Sphingolipid quantification was performed by tandem mass spectrometry using a triple quadrupole instrument (VG micromass model Quattro II, Waters) equipped with a nano electrospray source, as previously described (Jennemann et al., 2007;

Sandhoff et al., 2005). Equivalent lipid samples regarding the protein content were dissolved in 5 mM NH4OAc in CH3OH. Prior to being analyzed, internal standards were added to the aliquots.

Synthesized montanoyl- and melissoyl-CoA were also quantified by mass spectrometry. An equimolar mixture of 16:0, 18:0, 20:0, 24:0 and 26:0-CoA of known

concentration were used as internal standards to determine the concentration of the acyl-CoAs.

Sphingolipids, as well as fatty acids and acyl-CoAs were detected with precursor ion scans and neutral loss modus, respectively. The acquisition parameters and the internal standards used for quantification are compiled in the following table:

Lipid Scan

modus m/z Specific scan for

Collision

energy (eV) Internal standards

Acyl-CoA Neutral

All ESI-MS/MS measurements were performed by Prof. Dr. Roger Sandhoff from DKFZ in Heidelberg.

2.2.6.9. Thin layer chromatography (TLC)

Ceramide internal standards were analyzed and quantified by thin layer chromatography. After purification of internal standards with column chromatography, 20 µl of every third fraction of the eluate were spotted on double-sided HP-TLC silica plates (Silicagel 60 F254, Merck) using a Linomat IV (Camag). Separation was carried

out by running the TLC from both sides with CHCl3/CH3OH/glacial AcOH (188/11/1) in a horizontal chamber. Bands were then made visible by developing the TLC plate in 10% CuSO4 in 8% H3PO4 (10 min at 180 ºC). The fractions containing the purified ceramides were pooled and dried under a nitrogen stream.

For quantification, a dilution series of the purified products were spotted on a HP-TLC together with standards of known concentration and separated with CHCl3/CH3OH/glacial AcOH (188/11/1). After development with CuSO4 reagent, quantification of the bands was performed by densitometric scanning using a TLC scanner (Shimadzu CS-9301).

For epidermal extracts, lipids corresponding to 0.5 mg dry weight were spotted on a TLC plate and separated using a complex solvent system that included four different solvent mixtures. First, CHCl3/(CH3)2CO (1/1) was used to run the plate up to 4 cm of a 20 cm TLC plate, followed by CHCl3/CH3OH/glacial AcOH (60/35/8) run up to 7 cm, CHCl3/CH3OH/glacial AcOH (190/9/1) to 12 cm and finally n-hexane/methyl t-buthyl ether/glacial AcOH (90/10/1) up to the top. Lipids were then visualized using CuSO4 reagent.

Total mRNA expression analysis

2.2.7.1. Isolation of total RNA from tissue and eukaryotic cells

Total RNA was extracted with phenol, guanidinium thiocynate and chloroform according to Chomczynski and Sacchi, either with the ready-to-use Tryzol^ reagent or with a modified Chomczynski and Sacchi method (Chomczynski and Sacchi, 1987).

2.2.7.1.1. RNA isolation by modified CHOMCZYNSKI and SACCHI method

Juvenile mice testes (PN 5–25) were homogenized on ice in 3 ml of freshly prepared guanidinium thiocyanate buffer. Total RNA was then extracted with 300 µl of NaOAc (2 M, pH 4), 3 ml of Roti-Aqua-Phenol (Carl Roth), and 600 µl of CHCl3/isoamyl alcohol 24/1 (Sigma), with thorough mixing by inversion after the addition of every reagent. The suspension was incubated 15 min on ice, following by a centrifugation step at 15,000 × g for 30 min at 4 ºC. The upper aqueous phase containing the RNA was then transferred into a fresh tube, separated from the DNA and proteins present

in the lower phenol phase. Subsequently, 3 ml of isopropanol (100%) were added and RNA was precipitated overnight at – 20 ºC. A second precipitation step was performed by collecting the RNA pellet at 15,000 × g for 20 min, following the addition of 2 ml guanidinium thiocyanate buffer and 2 ml isopropanol, and overnight precipitation at – 20 ºC. After collection of RNA at 15,000 × g for 20 min, a washing step with 600 µl of 70% ethanol (– 20 ºC) was performed. Finally RNA was collected once more with 20 min centrifugation at 18,000 × g, and the pellet was then solubilized in ddH2O. Isolated RNA was flash frozen in liquid nitrogen and kept at – 80 ºC.

2.2.7.1.2. RNA isolation by Tryzol^ reagent

Tryzol^ reagent (Invitrogen), a ready-to-use monophasic solution of phenol and guanidinium thiocyanate, was used to isolate total RNA from cultured cells as well as from tissue biopsies (e.g. testes, skin), according to manufacturer’s instructions.

Cultured cells grown in monolayer in a 6-well plate were directly lysed with 1 ml of Tryzol^ reagent. For the isolation of RNA from tissue, biopsies were homogenized in 1 ml Tryzol^ per 50–100 mg of tissue. Skin biopsies were disrupted using a glass-teflon grinder (Glass-Cool^), while disruption of testes was performed using a power homogenizer (Ultra Turrax^ T25 basic). Sequentially, homogenates were incubated for 5 min at r.t. to enable complete cell lysis. RNA was extracted with the addition of 0.2 ml CHCl3 (per 1 ml of Tryzol^), followed by a thorough mixing, and an incubation step of 3 min at r.t. Following, phase separation was performed at 12,000 × g for 15 min, and the upper aqueous phase containing RNA was collected into a fresh tube. RNA precipitation was performed with 0.5 ml isopropanol, and RNA was pelleted at 12,000 × g for 10 min. To wash the RNA pellet, 1 ml ethanol (75%) was used, and RNA was finally collected at 7,500 × g for 5 min, and solubilized in DEPC or Braun water. All centrifugation steps were performed at 4 ºC.

2.2.7.2. DNAse digestion

Contaminating DNA was removed from RNA isolates using TURBO DNA-free Kit (Ambion). Thus, 10 µg of RNA dissolved in 15 µl of ddH2O were incubated for 30 min

at 37 ºC with 1 µl of DNase I and 1x DNase buffer. Following enzymatic treatment, 2 µl of DNase inactivation reagent were added, and after 2 min incubation at r.t., contaminants were pelleted at 10,000 × g for 2 min. Finally, purified RNA was transferred into a fresh tube.

2.2.7.3. RNA integrity assessment with RNA6000 nanochip

RNA integrity was analyzed by electrophoresis using an Agilent RNA 6000 nanochip according to the description of the manufacturer. The nanochip was evaluated with 2100 Bioanalyzer (Agilent Technologies) using the eukaryote total RNA assay. The RNA integrity number (RIN) was used to determine the quality of the isolated RNA.

RIN is an algorithm calculated by the software according to the complete electrophoretic trace of the RNA sample. Maximum RNA quality is defined by RIN values equal to 10, and ratios between 28S/18S of rRNA equal to 2.

2.2.7.4. Synthesis of double stranded cDNA

Reverse transcription of RNA into single stranded (ss) cDNA was performed with Superscript^ II. Briefly, 3 µg of isolated RNA diluted in 14 µl of ddH2O were mixed with 1 µl of oligo-dT12-18 primer (0.5 µg/µl) and 1 µl of dNTP mixture (10 mM). The samples were heated to 65 ºC for 5 min prior the addition of 4 µl of first strand buffer (5x) and 2 µl of DTT (100 mM). After incubating the samples for 2 min at 42 ºC, 1 µl of Superscript^ II (200 u/µl) was added, and reverse transcription was performed for 1 h at 42 ºC.

Sequentially, double stranded (ds) cDNA was synthesized from freshly prepared single stranded cDNA. To the ss cDNA was added 30 µl of second strand buffer (5x), 3 µl of dNTP mixture (10 mM), 4 µl DNA Polymerase I (10 u/µl), 1 µl of DNA ligase (10 u/µl), and 1 µl of RNAse H (2 u/µl) to a final volume of 153 µl with ddH2O. The reaction mixture was incubated for 2 h at 16 ºC. To assure blunt end of the ds cDNA termini, 2 µl of T4 DNA polymerase were added to the newly synthesized ds cDNA.

Afterwards, samples were incubated for 10 more min at 16 ºC in order to allow the enzymatic reaction of the polymerase. Finally, ds cDNA was purified using the QIAquick PCR purification kit (see 2.2.1.3) and eluted from the silica-based membrane with 50 µl of ddH2O.

All enzymes used for reverse transcription were purchased from Invitrogen.

2.2.7.5. Quantitative real time PCR (qRT-PCR)

Quantification of gene expression by real time PCR was performed using the LightCycler^ 2.0 system (Roche Diagnostics). For the amplification and detection of the gene of interest, the LightCycler^ FastStart DNA Master SYBR Green I was used in combination with specific primers for the target genes and 1:10 dilution of the synthesized cDNA. Generally, optimal amplification of target genes required divalent cations in a final concentration of 2.5 mM. The real time PCR reactions were set as follows:

The PCR reaction mixtures were loaded in LightCycler^ glass capillaries, and the qRT-PCR was performed immediately. A negative control was loaded in each PCR to assure no contamination of the samples. Amplicons of the target gene were generated during the 45 denaturation-annealing-elongation cycles (programs 2–4).

Following, melting curves of the amplified fragments were generated (programs 5–8) in order to evaluate the integrity of the products. Reactions were performed with biological triplicates and technical duplicates.

Primers were designed exon-spanning and localized at the 3’-prime region using Primer3 (v 0.4.0). Generally, amplicon size was between 50 and 200 bp, and the GC content was set to 45–55%. To exclusively enhance the target homologue gene, blast analysis and multiple sequence alignment (ClustalW, v 1.83) were performed to evaluate the homology of the primer with the other homologue family members. The sequence and annealing temperatures of the primers used in this study are listed in

section 2.1.12.3. Amplicons were further analyzed by agarose electrophoresis to assure the amplification of the appropriate products.

Quantification of the gene expression was calculated using the ∆∆CT method (Livak and Schmittgen, 2001), normalizing relative CT values to the expression of the housekeeping gene, Gapdh.

2.2.8. Animal experiments