Generation of GBA2 KO and GBA1/GBA2 DKO medaka
The ethics statement and maintenance of medaka were described previously (33). Medaka experiments were approved by the Animal Experiments Committee of Kyoto University and conducted in accordance with national guidelines. Medaka were maintained in an aquaculture system with recirculating water at 27 °C in a 14-h light/10-h dark cycle.
Medaka of the Kyoto-cab strain, a substrain of Cab, were used in this study. The generation and characterization of GBA1-deficient medaka were reported previously (33). GBA2-deficient medaka were generated using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, as reported previously (34). In brief, the cDNA sequence of medaka GBA2 was determined by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The medaka GBA2 gene consists of 18 exons encoding 858 amino acids. The crRNAs were designed using the CRISPR design tool (http://viewer.shigen.info/cgi-bin/crispr/crispr.cgi), and the following crRNA was used: 5’-GGAGGGCAAAGCACTGTCGGGGG-3. The crRNA and tracrRNA were constructed by Fasmac Co. (Kanagawa, Japan). The Cas9 RNA was synthesized from pCS2+hSpCas9 vector (Addgene #51815) using mMessage mMachine SP6 Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA mixture was injected into single-cell-stage embryos. The injected founders (F0) were raised to sexual maturity and back-crossed with wild-type (WT) to generate F1s. The GBA2 gene of F1s was sequenced, and novel heterozygous GBA2 mutation (GBA2+/−) medaka with 21 bases deleted and 2 bases inserted into exon 5 were obtained (Additional file 1: Fig. S1). These deletions and insertions resulted in a frame shift mutation, leading to the deficiency in protein expression and enzymatic activity of GBA2 in the brain. Off-target candidates were searched for using the Medaka pattern match tool (http://viewer.shigen.info/medakavw/crisprtool/). No alterations were found in three off-target candidates located on exons.
GBA2+/− medaka were back-crossed with WT medaka at least five times and then crossed with GBA1-deficient medaka to create GBA1/GBA2 DKO medaka. Medaka brains collected by surgery were directly snap-frozen in liquid nitrogen and then stored at −80 °C until use. The GBA2+/− medaka and GBA2 KO medaka were used in the previously reported study (35).
The GBA2 enzymatic activity assay
The measurement of GBA2 enzymatic activity was carried out as described previously (36, 37). Medaka brains were homogenized in 200 µl deionized water and centrifuged at 500 × g at 4 °C for 10 min. The supernatant was collected and centrifuged at 20,000 × g at 4 °C for 20 min. The pellet was rinsed with 200 µl of 50 mM potassium phosphate buffer, pH 5.8 and centrifuged at 20,000 × g at 4 °C for 15 min. This step was repeated twice. Next, 30 µl of 50 mM potassium phosphate buffer, pH 5.8 was added to the pellet, and the pellet was resuspended. The resulted suspension was used for the enzyme assay.
The reaction mixture contained 10 µl of the suspension and 20 µl of 4.5 mM 4-methlumbeliferyl β-d-glucopyranoside (Wako, #324-37411) in 100 mM citric acid and 200 mM disodium hydrogen phosphate buffer, pH 5.8. The reaction mixture was incubated at 37 °C for 60 min with or without 0.3 mM NB-DGJ (N-deoxygalactonojirimycin, #B690500; Toronto Research Chemicals Inc., Toronto, Canada). The reaction was terminated by adding 200 µl of 0.5 M sodium carbonate buffer at pH 10.7, and the fluorescence (excitation 55 nm and emission 460 nm) was measured by Fluoroskan Ascent FL (Thermo Fisher Scientific).
Locomotor function analysis
The medaka locomotor function analysis was performed as described previously (33). Medaka were transferred to a 20-cm-diameter tank filled with water to a depth of 2 cm at room temperature. After 5 min of rest, the free-swimming distance for 5 min was analyzed using an ethovision XT 5 software (Noldus, Leesburg, VA).
For the asyn, p62, LC3, neuron specific enolase (NSE) and β-actin analysis, the brains of medaka were homogenized in high-salt buffer containing 1% Triton X-100 (750 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 1% [v/v] Triton X-100, pH 7.5). For the GBA2 analysis, the brains of medaka were homogenized in RIPA buffer (50 mM Tris-HCl, 0.15 M NaCl, 1% [v/v] Triton X-100, 0.1% [w/v] sodium dodecyl sulfate (SDS), 1% [v/v] sodium deoxycholate, pH 7.5). The homogenate was centrifuged at 20,400 × g at 4 °C for 5 min. For SDS-soluble fractions, the pellet of asyn analysis was subsequently sonicated in SDS buffer (50 mM Tris-HCl, 2% SDS, pH 7.4) followed by centrifugation at 20,400 × g at 4 °C for 5 min. The supernatant was collected, and the protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). The supernatant was then mixed with sample buffer (1% [w/v] SDS, 12.5% [w/v] glycerol, 0.005% [w/v] bromophenol blue, 2.5% [v/v] 2-mercaptoethanol, 25 mM Tris-HCl, pH 6.6) and boiled at 95 °C for 10 min. The boiled samples containing 10 µg of protein or 0.75 µg for SDS-soluble fractions of protein were separated on 4-12% NuPAGE Bis-Tris Precast Gel (Thermo Fisher Scientific) or 10-20% SuperSep TMACE (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred to polyvinylidene difluoride membranes using a Trans-Blot SD semi Dry Transfer Cell (Bio-Rad Laboratories Inc., Hercules, California, USA).
To detect medaka asyn, the membranes were treated with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature before being blocked with 5% skim milk (33, 38). The following antibodies were used as the primary antibody: anti-β-actin (#A1978, 1:5000; Sigma-Aldrich Co., St. Louis, MO, USA), anti-LC3 (#PM036, 1:2000; MBL, Nagoya, Japan), anti-NSE (#M0873, 1:500; DAKO, Carpinteria, CA, USA), anti-medaka asyn (1:10,000) (33) and anti-p62 (#PM045, 1:500; MBL). Anti-medaka GBA2 antibody was raised against 377-395 amino acids of medaka GBA2 at Sigma-Aldrich Co. (1:1000). The membrane was incubated with anti-β-actin for 60 min at room temperature or with other primary antibodies for 1 day at 4 °C. Subsequent steps were performed according to the standard method using horseradish peroxidase-conjugated secondary antibodies (1:5000; Novus, Biologicals, Littleton, CO, USA) for 1 h at room temperature. The chemiluminescent signal was detected using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).
Confirmation of the cross-species reactivity of antibodies
To confirm the cross-species reactivity of the antibodies used in present study against medaka, the homology search for β-actin, LC3, NSE and p62 genes was performed (https://asia.ensembl.org/Oryzias_latipes/Info/Index), showing high homology of medaka β-actin, LC3, and NSE to the peptides used to raise each antibodies (93%, 93%, and 84%, respectively). Meanwhile, medaka p62 has only 45% homology to the peptide used to raise the p62 antibody. Therefore, we confirmed the cross-species reactivity of the antibody by overexpression of medaka p62 in cultured cells. The medaka p62 cDNA was cloned into pcDNA3 and expressed in HEK293T cells. A strong signal was detected at the molecular weight close to that of medaka p62 in the lanes of medaka p62-overexpressing HEK293T cells and GBA1 KO medaka brains, but not in the lane of mock transfected cells (Additional file 2: Fig. S2). The following is the brief description about this experiment. The sequence of medaka p62 cDNA was determined previously (33). The full-length medaka p62 cDNA was inserted into pcDNA3 to generate a medaka p62-expressing vector. Transfection of HEK293T cells (RIKEN Cell Bank, Tsukuba, Japan) with vector alone (pcDNA3-mock) or vector containing full-length medaka p62 (pcDNA3p62) was performed using polyethyleneimine MAX (#24765, Polysciences, Warrington, PA, USA) according to the manufacturer’s instructions. The cell lysates were obtained with RIPA buffer. The SDS-PAGE and chemiluminescent signal detection was performed as described above. The amount of loading protein was 5 μg for WT and GBA1 KO medaka brains and 0.05 μg and 0.25 μg for cell lysates, respectively.
Paraffin sections were used for immunohistochemical analysis as reported previously (33). Medaka brains were collected by surgery. The collected brains were fixed with 4% (w/v) paraformaldehyde in PBS at 4 °C for 1 day and stored in 70% ethanol until use. The fixed samples were dehydrated and embedded in paraffin using Surgipath FSC22 (Leica, Wetzlar, Germany), and sections were acquired using a Microm HM 325 (Thermo Fisher Scientific). The thickness of the sections was set at 20 µm for tyrosine hydroxylase (TH) -positive cell counting and 8 µm for other analysis. The following antibodies were used as the primary antibody: anti-TH (#MAB318, 1:1000; Merck Millipore, Burlington, MA) and anti-medaka asyn (1:2000) (33). The sections were incubated with the primary antibody at 4 °C for 1 day after blocking with 4% skim milk. Histofine (#414322; Nichirei Bioscience Inc., Tokyo, Japan) was used as the secondary antibody for diaminobenzidine staining.
The number of dopaminergic (DA) neurons in the middle diencephalon and noradrenergic (NA) neurons in the locus coeruleus were counted as previously described (33). The numbers of TH-positive neurons with visible nuclei were counted under the microscope (Cx41; Olympus, Tokyo, Japan).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
RNA was isolated from medaka brains using Qiazol (QIAGEN). cDNA was synthesized using the PrimeScript RT reagent kit of Perfect Real Time (#RR037A; Takara, Kyoto, Japan). The quantification of cDNA was performed with the LightCycler 480 using LightCycler 480 SYBR GreenⅠ Master (#04887352001; Roche Diagnostics, Mannheim, Germany). The following primer sets were used: TNF-α: 5’-ATTGGAGTGAAAGGCCAGAA-3’ and 5’-ACTAATTTGAGACCGCCACG-3’; β-actin: 5’-TCCACCTTCCAGCAGATGTG-3’ and 5’-AGCATTTGCGGTGGACGAT-3’; apolipoprotein E (ApoE)-b: 5’-GACGAGAGTTGGAGACCCTGA-3’ and 5’-ACTGGTGCTTGTGGTGATGG-3’; and asyn: 5’-ATGGACGCGTTAATGAAGGGTTT-3’ and 5’-TCAGTCATCGCTGTCTTCCT -3’.
High performance liquid chromatography for the dopamine quantification
Medaka brains were homogenized in 100 µl of 0.1 M HClO4 containing 4mM Na2S2O5 and 4 mM diethylenetriaminepentaacetic acid. The supernatant by centrifugation at 20,400 × g for 5 min was used for measurement. High performance liquid chromatography (HPLC) was conducted with a mobile phase A (acetonitrile:methanol, 1000:25.9:62.9 [v/v/v], with 0.1 M phosphate, 0.05 M citrate, 4 mM sodium 1-heptanesulfonate and 0.1 mM EDTA, pH 3.0). Dopamine was detected with series coulometric detector (ESA, Inc., Chelmsford, MA, USA). Data were collected and analyzed on a CHROMELEONTM Chromatography Data Systems 6.40 (Dionex, Sunnyvale, CA, USA).
The material for the sphingolipid profile analysis
β-d-Glucopyranosyl-(1→1)-N-lauroyl-d-erythro-sphingosine (GlcCer [d18:1-C12:0]), β-d-glucopyranosyl-(1→1)-d-erythro-sphingosine-d5 (GlcSph-d5), N-lauroyl-d-erythro-sphingosine (ceramide [d18:1-C12:0]) and d-erythro-sphingosine (C17 base) (d17:1-sphingosine) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). For liquid chromatography (LC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS), high-performance LC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Waltham, MA, USA), chloroform and distilled water were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), and ammonium formate was purchased from Sigma Aldrich, Japan.
Lipid extraction for the sphingolipid profile analysis
The frozen tissue (approximately 5 mg) was homogenized, and total lipids were extracted with a chloroform:methanol (C:M) (2:1 [v/v], 5 ml) mixture spiked with 1 pmol/mg frozen tissue of GlcCer (d18:1-C12:0), GlcSph-d5, ceramide (d18:1-C12:0) and d17:1-sphingosine as internal standards. Extracts were dried under a flow of N2 gas and hydrolyzed for 2 h at room temperature in C:M (2:1 [v/v], 2 ml) containing 0.1 M KOH. The reaction mixture was neutralized with 7.5 µl of glacial acetic acid.
For the analysis of GlcCer, galactosylceramide (GalCer), GlcSph and galactosylsphingosine (GalSph: psychosine), the neutralized reaction mixture was subjected to Folch’s partition, and the lower phase was dried under a flow of N2 gas. For the analysis of ceramide and sphingosine, the neutralized reaction mixture was dried under a flow of N2 gas. The resulting lipid films were suspended in C:M (2:1, v/v) at a concentration of 100 µg frozen tissue/µl, and aliquots were subjected to LC-ESI-MS/MS.
LC-ESI-MS/MS for the sphingolipid profile analysis
LC-ESI-MS/MS was performed on an LC system Nexera X2 (SHIMADZU, Kyoto, Japan) attached to a triple-quadrupole linear ion trap mass spectrometer (QTRAP4500; SCIEX, Tokyo, Japan). The LC-ESI-MS/MS datasets were analyzed with the MultiQuant™️ (ver. 2.1) and Analyst (SCIEX) software programs. Target lipids were monitored in multiple reaction monitoring (MRM) mode using specific precursor-product ion pairs, as detailed in Additional file 3: Table S1. Peak areas were integrated and quantified relative to the associated internal standard.
GlcCer, GalCer, GlcSph and GalSph were analyzed as previously reported (15, 39, 40) by hydrophilic interaction chromatography (HILIC)-ESI-MS/MS with minor modifications. In brief, 100 µg frozen tissue/µl of the lipid extracts was diluted 10-fold with mobile phase A (acetonitrile:methanol:formic acid, 97:2:1 [v/v/v], with 5 mM ammonium formate), and aliquots (10 µl) were applied to an Atlantis silica HILIC column (2.1 mm i.d.×150 mm, particle size, 3 µm; Waters, Milford, MA) maintained at 40 °C. Samples were eluted at a flow rate of 0.15 ml/min using the following gradient of mobile phase B (methanol:water:formic acid, 89:9:1 [v/v/v], with 20 mM ammonium formate): 3.3 min, 0%; 13.4 min, 0%-35% linear gradient; 1.3 min, 35%-70% linear gradient; 3 min, 70% (washing step); 29 min, 0%, flow rate increased to 0.2 ml/min (equilibration); 1 min, 0%, flow rate decreased to 0.15 ml/min. The mass spectrometer was set to positive ion mode (ion spray voltage, 5500 V; curtain gas pressure, 30 psi; nebulizer gas pressure, 90 psi; heating gas pressure, 30 psi, temperature, 100 °C) using MRM detection for a targeted analysis. The quantitative values of GlcCer and GalCer with various chain lengths of fatty acids (C14:0, C16:0, C18:1, C18:0, C20:1, C20:0, C22:1, C22:0, C23:1, C23:0, C24:1, C24:0 and C26:1) were summarized.
Ceramide and d-erythro-sphingosine (C18 base) (sphingosine (C18 base)) were analyzed as previously reported (39) by reversed-phase liquid chromatography (RPLC)-ESI-MS/MS with minor modifications. The lipid extracts dissolved in C:M (2:1, v/v) were diluted 10-fold with mobile phase B (M:W 85:15 [v/v], 5 mM ammonium acetate) and applied to an RP column (Luna C18(2) column; 2 mm i.d.×250 mm, particle size, 3 μm; Phenomenex, Torrance, CA, USA) maintained at 36 °C and at a flow rate of 0.15 ml/min. The samples were then eluted with the following gradients of mobile phase A (methanol pure, 5 mM ammonium acetate): 2 min, 0%; 13 min, 0%-100% linear gradient; 40 min, 100% (washing step); 15 min 0% (equilibration). The mass spectrometer was set to positive ion mode (ion spray voltage, 5500 V; curtain gas pressure, 20 psi; nebulizer gas pressure, 80 psi; heating gas pressure, 40 psi, temperature, 100 °C), utilizing either MRM detection for a targeted analysis. The quantitative values of ceramide with various chain lengths of fatty acids (C16:0, C18:0, C24:1 and C24:0) were summarized.
For comparison of two groups, at wo-tailed unpaired Student’s t-test was performed. An F test was performed to evaluate the differences in variances. For comparison of three or more groups, one-way ANOVA with Tukey’s post-hoc test or Newman-Keuls multiple comparison test was performed. A Brown–Forsythe test was performed to evaluate the differences in variances. Differences with p values of less than 0.05 were considered significant. Statistical calculations were performed with GraphPad Prism Software (GraphPad) Version5.0 and 7.04.