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Mar 2017
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Generation, Analyzing and in-vivo Drug Treatment of Drosophila Models with IBMPFD
果蝇IBMPFD模型的建立、分析及体内药物治疗   

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Abstract

Missense mutations of p97/cdc48/Valosin-containing protein (VCP) cause inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative diseases. The pathological mechanism of IBMPFD is not clear and there is no treatment. We generated Drosophila models of IBMPFD in adult flight muscle in vivo. Here we describe a variety of assays to characterize disease pathology and dissect disease mechanism, and the consequences of in vivo feeding of VCP inhibitors.

Keywords: Drosophila (果蝇), Inclusion body myopathy (包涵体肌病), Paget disease and frontotemporal dementia (IBMPFD) (佩吉特病和额颞痴呆(IBMPFD)), VCP/p97 (VCP/p97), Mitochondria (线粒体), Muscle (肌肉), Disease models (疾病模型), Inhibitors (抑制剂), Drug treatment (药物治疗)

Background

Mutations of VCP/p97 cause inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD), a degenerative disease in multiple systems including the brain, muscles and bones in an autosomal dominant fashion (Watts et al., 2004). Mutations in VCP are also associated with 1-2% of cases of sporadic amyotrophic lateral sclerosis (ALS), as well as hereditary spastic paraplegia and Charcot-Marie-Tooth 2 neuropathy (Abramzon et al., 2012; de Bot et al., 2012; Gonzalez et al., 2014). The R155H mutation is the most frequently identified in patients, while individuals with the A232E mutation have the most severe clinical manifestation (Kimonis et al., 2008a; Ritson et al., 2010). 90% of IBMPFD patients display myopathy, frequently the earliest symptom (Weihl et al., 2009). 50% of patients will develop Paget’s disease of bone, affecting skull, spine, hips and long bones. One-third of the patients develop frontotemporal dementia (Kimonis et al., 2008b; Weihl et al., 2009). Patients ultimately develop cardiopulmonary failure (Kimonis et al., 2008b; Weihl et al., 2009). VCP encodes a highly conserved and abundant AAA+ ATPase which participates in multiple cellular processes (Meyer et al., 2012). Since VCP assembles as a hexamer, it has been controversial whether disease mutants with increased ATPase activity cause disease through a dominant-active (Chang et al., 2011) or dominant-negative mechanism (Ju et al., 2009; Ritz et al., 2011; Bartolome et al., 2013; Kim et al., 2013; Kimura et al., 2013). We built in vivo IBMPFD models to understand the pathogenesis of the disease and find potential treatments.

The specific assays for IBMPFD disease in flies include in situ cell death detection, muscle protein Western blot assays, immunofluorescence staining of disease markers, muscle integrity assay/Toluidine Blue staining, and muscle mitochondrial ultrastructural studies/Electron Microscopy (EM) imaging, and in vivo VCP inhibitors treatment (Yun et al., 2008; Zhang et al., 2017; Ma et al., 2018).

Materials and Reagents

  1. Razor blade (VWR North American, catalog number: 55411-050 )
  2. Microcentrifudge tube (Denville Scientific Inc., catalog number: C2171 )
  3. PCR tube (USA Scientific)
  4. FORMVAR FILM on 100 Square Mesh Copper Grid (Electronic Microscopy Sciences, catalog number: FF100-Cu )
  5. FORMVAR CARBON FILM on 2 x 1 mm oval slot Copper Grid (Electronic Microscopy Sciences, catalog number: FCF2010-Cu )
  6. Disposable pellet pestle (Kimble, catalog number: 749521-0500 )
  7. PCR tube
  8. Dissection dish
  9. Needles
  10. Pipette tips
  11. Toothpick (Fisher Scientific Education, S04180 )
  12. 0.22 µm filter (Millipore, MillexGP Filter Unit)
  13. Aluminum foil (Fisher Scientific, catalog number: 01-213-101 )
  14. Immobilon-P PVDF transfer membrane (Millipore, catalog number: IPVH00010 )
  15. Drosophila strains: UASt-VCP WT, VCP RH and AE lines were gifts from Dr. Tzu Kang Sang (Chang et al., 2011)
  16. Drosophila Strain
    IFM-Gal4, with Gal4 under the control of the indirect flight muscle promoter, derived from the flightin gene (Yun et al., 2014). The IFM promoter provides a strong pulse of expression in late pupal stages and the first few days of adulthood, and is thereafter silent (Kandul et al., 2016). Drosophila strains were maintained in a 25 °C humidified incubator.
  17. 20% paraformaldehyde, EM Grade (Electronic Microscopy Sciences, catalog number: 15713S )
  18. 8% glutaraldehyde solution, EM Grade (Electronic Microscopy Sciences, catalog number: 16020 )
  19. 16% paraformaldehyde aqueous solution, EM Grade (Electronic Microscopy Sciences, catalog number: 15710 )
  20. Schneider’s Buffer (GIBCO, catalog number: 21720-024 )
  21. In Situ Cell Death Detection Kit (Roche, catalog number: 11684795910 )
  22. Rhodamine Phalloidin (Life Technology, catalog number: R415 )
  23. Bovine Serum Albumin (Fisher Bio Reagent, catalog number: BP1600-100 )
  24. Fluoromount-G (Southern Biotech, catalog number: 0100-01 )
  25. Pierce RIPA lysate Buffer (Thermo Scientific, catalog number: 89900 )
  26. Pierce Protease Inhibitor Tablets EDTA free (Thermo Scientific, catalog number: 88266 )
  27. Laemmli SDS Sample Buffer, 6x (Bioland Scientific LLC, catalog number: SAB02-02 )
  28. Anti-mouse IgG horseradish peroxidase linked whole antibody (from sheep) (GE Healthcare, catalog number: NXA931 )
  29. Anti-rabbit IgG horseradish peroxidase linked F(ab’)2 fragments (from donkey) (GE Healthcare catalog number: NA9340V )
  30. Goat anti-rabbit/mouse Alexa Fluor 546/488 secondary antibody (Life Technologies, catalog numbers: A11034 / A11029 / A11035 / A10036 )
  31. Tween-20 500 ml (Hoefer, catalog number: 56-40-6 )
  32. Immobilon Western Chemiluminescent HRP Substrate (Millipore, catalog number: WBKLS0500 ).
  33. Embed812 (Electronic Microscopy Sciences, catalog number: 14900 )
  34. Osmium Teroxide (OsO4) 4% aqueous solution (Electronic Microscopy Sciences, catalog number: 19150 )
  35. DDSA (dodecenyl succinic anhydride) (Electronic Microscopy Sciences, catalog number: 13710 )
  36. NMA (nadic methyl anhydrate) (Electronic Microscopy Sciences, catalog number: 19000 )
  37. BDMA (benzyldimethylamine) (Electronic Microscopy Sciences, catalog number: 11400 )
  38. Propylene Oxide (Electronic Microscopy Sciences, catalog number: 20401 )
  39. Toluidine Blue O Powder (Electronic Microscopy Sciences, catalog number: 22050 )
  40. Sodium Borate (Fisher Scientific, catalog number: S249-500 )
  41. Uranyl Acetate Dihydrate (Ted Pella, catalog number: 19481 )
  42. Lead Nitrate (Sigma-Aldrich, catalog number: 22862-100G )
  43. Sodium Citrate, dihydrate (EMD, catalog number: SX0442-1 )
  44. Sodium Hydroxide Certified ACS Pellets NaOH (Fisher Scientific, catalog number: S318-500 )
  45. NMS-873 (3-[3-(cyclopentylthio)-5-[[[2-methyl-4'-(methylsulfonyl) [1,1'-biphenyl]-4-yl] oxy] methyl]-4H-1,2,4-triazol-4-yl]-pyridine (Selleckchem, catalog number: S7285 )
  46. ML240,2-(2-Amino-1H-benzimidazol-1-yl)-8-methoxy-N-(phenylmethyl)-4-quinazolinamine (Sigma-Aldrich, catalog number: 1346527-98-7 )
  47. Permount mounting medium (Fisher Scientific, catalog number: SP15-100 )
  48. 0.1% Triton X-100
  49. PBS
  50. TUNEL Enzyme
  51. β-mercaptoethanol
  52. Ethanol
  53. Muscle Dissection Fixative Buffer (see Recipes)
  54. Dissection Buffer (see Recipes)
  55. TUNEL Blocking Buffer (see Recipes)
  56. 0.2 M Phosphate Buffer (pH = 7.4) (see Recipes)
  57. 10 ml EM Fixative Buffer (see Recipes)
  58. Epon Mix (Medium) (see Recipes)
  59. 1% Toluidine Blue Staining Solution (see Recipes)
  60. Uranium Acetate Staining Solution (see Recipes)
  61. Lead citrate Staining Solution (see Recipes)

Equipment

  1. Pipette (Denville Scientific Inc., models: P10, P20, P200, P1000)
  2. Diamond knife Ultra 45° (Diatome, catalog number: MX5341 )
  3. PELCO Reverse self-closing Tweezers (Ted Pella, catalog numbers: 5373/ 5375-NM )
  4. DUMONT Biology Grade Tweezers (Ted Pella, catalog numbers: 505/505-U )
  5. Pellet pestle motor (Kimble/Kontes, catalog number: 749540-0000 )
  6. JEOL 100CX transmission electron microscope (UCLA Brain Research Institute, Electronic Microscopic Core Facility)
  7. Ultracut ultramicrotome (Leica EM UC6, Dr. Frank Laski, UCLA Molecular Cellular and Development Biology)
  8. PELCO Pro Reverse (self-closing) tweezers (Ted Pella, catalog number: 5375-NM )
  9. Dissection tweezers (Dumont Biology Switzerland, Electronic Microscopic Science)
  10. Diamond knife (DiATOME, Ultra 45° MX5341 )
  11. Formvar mesh/slot grid (Electronic Microscopic Science, catalog numbers: FCF2010-Cu , FF100-Cu -50)
  12. Water bath (Fisher Scientific, model: Isotemp )
  13. The Mini-PROTEAN® tetra handcast systems and tetra blotting bodule (Bio-Rad)
  14. Safety fume hood (UCLA facility 4315-3309-2)
  15. Heat block (Fisher Scientific, model: Isotemp )

Procedure

  1. Indirect flight muscle dissection
    1. Anesthetize flies on a CO2 plate. Cut the head and abdomen off using a razor blade.
    2. Fix the thoraces in 4% paraformaldehyde/Schneider’s Buffer for 45 min at room temperature in either a microcentrifuge tube or PCR tube.
    3. Transfer the post-fix thoraces into a dissection dish containing 0.1% Triton X-100/PBS. Triton X-100 helps to break the surface tension over the cuticles and facilitates muscle piece dissection in the following steps.
    4. Indirect flight muscles (IFM, indicated by red arrows in Figures 1A and 1B) are located in the middle of the thoraces. IFMs can be dissected out using either 30-gauge needles or tweezers. Use needles or tweezer to gently open up the thorax from either the dorsal or ventral midline and expose the IFM. The IFMs are two large groups of muscles that run anterior-posterior in the middle of the thorax (Figure 1C). Each group of IFMs contains 5 large muscles that lie parallel to each other and are located at the dorsal surface of the half-dissected thorax. Carefully isolate each muscle by cutting at the ends that attach the muscle to the cuticle. One well-fixed wildtype thorax can generate 5-10 fragments of intact muscle (Figure 1C). Flies that carry an IFM targeted GFP marker can be used to further confirm that the correct muscle has been dissected (Figure 1C).


      Figure 1. Indirect Flight Muscle (IFM) Dissection. A. The location of IFM in intact thorax (red arrows). B. The sagittal plane of a fixed thorax, red arrow indicating where the IFM are located. C. Dissected muscle pieces in the dissection dish. The genotype of the flies used is IFM-Gal4>UASt-mitoGFP. Scale bar: 0.5 mm.

  2. In situ cell death detection/TUNEL Assay for IBMPFD flies
    1. Fix the thoraces and dissect the IFMs from WT, VCP WT, RH and AE flies as described above and collect into PCR tubes.
    2. Introduce TUNEL Blocking buffer into the tubes containing muscle for 30 min. The TUNEL Blocking buffer contains 0.2% Triton X-100 that permeabilizes and blocks the muscle sample at the same time.
    3. Add TUNEL Enzyme (4 µl) and 10x Reaction Buffer (36 µl) from the in-situ Cell Death Detection Kit and incubate for 2-3 h at 37 °C in a water bath. Use a pipette to gently mix the sample every 30 min.
    4. Wash the samples with PBS twice.
    5. Replace PBS with Fluoromount-G mounting solution. Mount IFMs on a microscopic slide using either a glass pipette or 200 µl pipette tip and visualize the muscle with a confocal microscope using an excitation wavelength of 546 nm. In 6 days old flies, VCP RH and AE expression leads to significant cell death (Zhang et al., 2017). This is observed as extensive red nuclear staining.

  3. Immunofluorescence staining and confocal microscopy imaging
    1. Dissect the fixed muscle as described above. Then wash with PBS and permeabilize muscles with 0.2% Triton X-100/PBS for 3-4 h on the rocker at room temperature followed by 5 min wash with 1x PBS.
    2. For myofibril staining, add Rhodamine Phalloidin (1:500 dilution) and stain for 2 h at room temperature or 4 °C overnight.
    3. For immunofluorescence staining, fixed muscle fragments are incubated with primary antibody diluted to the desired concentration (1:100 or 1:200) in 0.2% Triton X-100/PBS at 4 °C overnight. We used anti-TARTAR Binding Protein or anti-TDP43 at a concentration of 1:100.
    4. Wash 3x each for 10 min with 0.2% Triton X-100/PBS.
    5. Incubate muscle fragments with a goat anti-rabbit/mouse Alexa Fluor 488 or 546 secondary antibody (1:200 in 0.2% Triton X-100/PBS) for 2 h at room temperature or 4 °C overnight.
    6. Wash three times (10 min each time) with 0.2% Triton X-100/PBS and mount samples on a microscope slide with Fluoromount-G. Samples are visualized under a confocal microscope with an excitation wavelength at 488 nm or 546 nm corresponding to the secondary antibodies. In 6-day old flies, TDP43 localizes both in the nucleus and sarcoplasmic/cytosolic area of the IFM in wildtype flies; the nucleus localization pattern is lost and more aggregates are observed in the sarcoplasmic/cytosolic region in VCP RH and AE mutants (Zhang et al., 2017).

  4. Protein lysates and Western blot
    1. Anesthetize flies with CO2, isolate thoraces as above, and put them in a microcentrifuge tube on ice.
      Note: The thorax is not fixed.
    2. Add RIPA lysis buffer with protease inhibitors, 10-15 µl for each thorax, 5-10 thoraces for each genotype.
    3. Homogenize the thoraces in the lysis buffer with prechilled the polypropylene pellet pestles 5-6 times, followed by spinning the pestle with the mortar at the highest speed for 10-15 s. Incubate lysate on ice for 30 min to fully lyse the tissue.
    4. Centrifuge protein lysates at 10,000 x g for 15 min and transfer the supernatant to a clean microcentrifuge tube.
    5. Boil the supernatant with 6x SDS sample buffer with β-mercaptoethanol at 95 °C for 5 min.
    6. Load proteins and let them separate in 8% SDS-PAGE Gels in Tris/SDS running buffer and run it at 80 volts.
    7. Transfer proteins to PVDF membrane for 2 h in Tris/Glycine Buffer with 15% methanol.
    8. Incubate transfer membrane with 3% BSA/PBS for 1 h.
    9. Incubate the membrane with primary antibody diluted in 1% BSA/0.01% Tween-PBS at 4 °C overnight.
    10. Wash the membrane in 0.01% Tween-PBS for three times (10 min each) and incubate with anti-mouse/rabbit IgG HRP linked secondary antibodies diluted in 5% non-fat milk/0.01% Tween-PBS for 2 h at room temperature.
    11. Develop the membrane with Immobilon Western Chemiluminescent HRP Substrate Kit. We expressed VCP WT, VCP RH and AE at comparable levels in the thoraces (Zhang et al., 2017).

  5. Toluidine blue staining and electronic microscopy for IBMPFD flies
    Fixation and Embedding
    Note: Perform Step E4 and subsequent steps in a safety fume hood.
    1. Anesthetize flies and cut the thoraces, quickly dip in 95% ethanol (this helps to break the surface tension so as to facilitate subsequent immersion in the EM fixative buffer). Then transfer thoraces to 0.5 ml ice-old EM Fixative Buffer (below) in a microcentrifuge tube.
    2. Fix for a minimum of 2 h on ice with rocking. If fixation is carried out for longer or the protocol needs to be paused, samples may be left in fixative after the 2 h time point and placed at 4 °C overnight or until ready to proceed. Thoraces will sink to the bottom of microcentrifuge tube.
    3. Rinse each sample with 0.1 M phosphate buffer for three times (10 min each time) at room temperature.
      Note: All subsequent steps should be carried out in a Bio-safety hood.
    4. Post-Fix samples in 1% OsO4 in ddH2O (freshly prepared) for 2 h at room temperature. Samples should not be fixed with OsO4 for more than 2 h or the samples will become brittle.
    5. Rinse sample with ddH2O for three times (10 min each) at room temperature.
    6. Dehydrate samples in 70% and 95% ethanol for 5 min each at room temperature.
    7. Dehydrate samples in 100% ethanol twice (10 min each) at room temperature.
    8. Dehydrate samples in 100% propylene oxide twice (7 min each) at room temperature.
    9. Infiltrate samples in 1:1 mixture of 100% propylene oxide and Epon mix for 30 min at room temperature.
    10. Incubate samples in 100% Epon Mix (1.5% BDMA added) overnight at room temperature.
    11. Introduce samples in 100% Epon Mix (1.5% BDMA added) into an embedding mold. In the bottom right of each well, put a piece of paper with sample name/number (Figure 2A).
    12. Polymerize Epon at 60-70 °C overnight.


      Figure 2. Drosophila thorax embedding and section position. A. Two individual samples embedded and blocked with labels. B. Properly embedded fly thorax (red arrow) in the Epon Resin. The sagittal plane of the thorax (white dotted lines) should be parallel to the edge of the resin block so as to facilitate the section. C. A well-trimmed resin block with a thorax sample after thick section. Glass knife or Diamond knife blades should run parallel to the sagittal plane of the thorax. Scale bar: 1 mm.

    Sectioning and Staining
    1. Toluidine Blue Staining
      1. Well-embedded thorax samples are positioned in the resin block as shown (Figure 2B). The sagittal midline of the thorax is parallel to the edge of the resin block.
      2. Use a razor blade to cut away the Epon around the sample as much as possible (Figure 2C). Decreasing the section surface will increase the section quality.
      3. Cut thick sections (1.5-2 µm) for Toluidine Blue staining and to determine the area within which thin sections will be taken for electron microscopy imaging.
        1. Use a microtome with glass knife to cut thick sections. Transfer sections to water drops on the glass slide using a toothpick.
        2. Evaporate the water by putting the slide on a 70 °C heat block. This will flatten the section samples.
        3. Apply 1% Toluidine Blue to fully cover the section sample area and put the slide on the 70 °C heat block until the edge of the Toluidine Blue liquid starts to dry (2-5 min). Do not over dry the Toluidine Blue solution or the section will become contaminated by the dry solute in the staining buffer, which results in degradation of image quality.
        4. Rinse the slide in ddH2O until non-bound stain is removed.
        5. Let the section dry completely before applying the Permount mounting solution.
      4. A well-aligned thick section sample of a wildtype thorax is shown in Figure 3A (Indirect flight muscle pieces can be easily observed). Higher magnification images of the tissue samples can be utilized (Figure 3B) to assay muscle tissue integrity. In 6-day old flies, VCP RH and AE expressing flies have disrupted muscle tissue integrity as compared to WT and VCP WT controls (Zhang et al., 2017).


        Figure 3. Toluidine Blue and EM images of IFM. A-B. Toluidine Blue staining of thick sections of thorax at different magnifications. C. EM image of a thin section of wildtype thorax. D. Mitochondrial size quantification using ImageJ. Scale bar: 1 µm (119 pixels). Cross-section area of selected mitochondrion is 2.92 µm2 (Red square).

    2. Electronic microscopy sample preparation
      1. Select well-aligned samples from the Toluidine Blue staining to be used in thin sectioning (80-90 nm).
        1. Use a microtome with a Diamond knife (with sterilized ddH2O) to cut thin sections. The sections should appear silver to gold color.
        2. Use a fine needle to position 1-4 sections in a line on the water surface for grid mounting.
        3. Pick up an empty slot grid that is not coated with formvar with tweezers. Gently touch the grid to sections floating on the surface. The sections will attach to the middle of the slot within the water drop.
        4. Use a pair of self-closing tweezers to hold a new formvar-sealed slot grid or mesh grid with the shiny-side up. Place the section-attached slot grid onto the shiny side of the new formvar grid.
        5. Remove the water drop between two grids by placing the sharp corner of a triangle-shaped filter paper at the edge between the grids. Sections will attach to the formvar-sealed new grids and flatten.
        6. Remove the empty grid and reuse it to pick up additional sections. The section-attached grid can be stored in the grid box for further staining as described below.
      2. Prepare the uranium acetate (UA) solution and filter through a 0.22 µm filter system. Float the section side of the grid on the UA solution for 15 min at room temperature. Wash the grid five times (1 min each) in a ddH2O water drop or three times (1 min each) in 3 separate beakers containing ddH2O.
      3. Put the grid section with shiny side down on a drop of the lead citrate solution and stain for 5-10 min at room temperature. Wear a mask to prevent breathing on to the sections as excess CO2 will affect staining quality. Keep the staining dishes covered with the plastic cover that comes with the staining dish.
      4. Wash the grid with ddH2O as above.
      5. Transfer the stained grid to the grid box for further imaging.
      6. Figure 3C shows an image of wildtype muscle visualized using TEM at 10,000x. In 6-day old flies, VCP RH and AE expressing flies display disrupted actin and mitochondrial structure as compared with wildtype and VCP WT flies (Zhang et al., 2017).
    3. Mitochondrial cross-section size quantification
      1. Import an ultrastructural electron microscopy image (10,000x magnification) into ImageJ Software (National Institute of Health), Figure 3D.
      2. After setting the scale (analyze>set scale: 119 pixels = 1 µm), using Polygon selection (Yellow line) trace out each mitochondrion on the image and measure its area (analyze > measure), Figure 3D. All the mitochondria on the image are individually measured.
      3. At least three images are analyzed for each thorax and 3 thoraxes of each genotype were examined.
      4. An independent Student’s t-test is used to test for statistical significance between different genotypes.

  6. In vivo VCP inhibitor treatment
    1. Dissolve powdered forms of NMS-873 (19.2 mM, stock concentration) and ML240 (25.2 mM, stock concentration) in DMSO as stocks.
    2. Dilute Stock solution in ethanol/ddH2O (100 µl Ethanol + 600 µl ddH2O) to the desired concentration. Ethanol can help keep the compound in solution. Heat Drosophila food in a microwave for 20 s until it fully melts, and then let it cool down to < 50 °C. Add the chemical in DMSO/ethanol H2O to the food along with 10 µl of food dye per 4 ml of fly food, and hand-mix it until the color appears homogenous. Vials with cotton plugs can be dried at room temperature if food seems wet. DMSO/ethanol in comparable amounts is used as a vehicle control.
    3. Put Drosophila parents of desired genotypes in food containing DMSO/ethanol or inhibitors for 3 days to lay eggs and then remove them. Progeny growth then occurs in the presence of vehicle control or test compound.
    4. Immediately after eclosion of adults (hatch from their pupal case), transfer these adults to freshly prepared food containing the same concentration of DMSO or inhibitor, and feed the flies for the desired number of days before assaying them.
    5. VCP inhibitors feeding significantly reversed the muscle disintegration, muscle cell death and ultrastructural mitochondrial defects in VCP RH and AE flies (Zhang et al., 2017).

Recipes

  1. Muscle Dissection Fixative Buffer (500 µl)
    100 µl 20% paraformaldehyde
    400 µl Schneider’s Buffer
    Store at 4 °C
    Note: Preferably prepare fresh each experiment.
  2. Dissection Buffer
    0.1% Triton X-100
    1x PBS Buffer
    Store at room temperature
  3. TUNEL Blocking Buffer
    50 mM Tris-Cl (pH = 7.4)
    188 mM NaCl
    0.2% Triton X-100
    1% BSA
    Store at 4 °C
  4. 0.2 M Phosphate Buffer (pH = 7.4)
    Solution X: 3.516 g Na2HPO4·2H2O/100 ml ddH2O
    Solution Y: 2.76 g NaH2PO4·H2O/100 ml ddH2O
    Mix 40.5 ml of Solution X with 9.5 ml of Solution Y, then get 50 ml of 0.2 M Phosphate Buffer. The pH should be 7.4
    Store at room temperature
  5. 10 ml EM Fixative Buffer
    1.25 ml 8% glutaraldehyde
    0.625 ml 16% paraformaldehyde
    5.0 ml 0.2 M phosphate buffer
    3.125 ml ddH2O Sterile
    Store at 4 °C
  6. Epon Mix (Medium)
    Embed812                                                     20 ml
    DDSA (dodecenyl succinic anhydride)      16 ml
    NMA (nadic methyl anhydrate)                  8 ml
    BDMA (benzyldimethylamine)                   0.9 ml (Add fresh for each embedding)
    Mix thoroughly at least 1 h before embedding
    Can be stored at room temperature in light-proof container. Preferably prepare fresh each time
  7. 1% Toluidine Blue Staining Solution
    1 g Toluidine blue powder
    1 g Sodium Borate
    100 ml of ddH2O
    0.22 µm filtered before use.
    Store at room temperature in light-proof container
  8. Uranium Acetate Staining Solution
    4 g Uranyl acetate
    100 ml ddH2O
    Dissolve fully (heat up to 70 °C to facilitate dissolving)
    0.22 µm filtered before use
    Store at room temperature in light-proof acrylic storage container that protects from beta radiation
  9. Lead Citrate Staining Solution
    Boil 100 ml ddH2O to get rid of CO2 in a small beaker covered with aluminum foil let it cool to room temperature
    1.33 g lead nitrate
    1.76 g sodium citrate
    30 ml CO2-free ddH2O
    8 ml 1 N NaOH (4 g NaOH in 100 ml ddH2O)
    Add CO2-free ddH2O to a total volume of 50 ml
    Store at 4 °C in light-proof container

Acknowledgments

We are grateful to the generous support from the National Institute of Health (National Institute on Aging), Glenn Foundation for Medical Research, the Natalie R. and Eugene S. Jones Fund in Aging and Neurodegenerative Disease Research, Kenneth Glenn Family Foundation, funds from the UCLA Laurie and Steven Gordon Commitment to Cure Parkinson’s Disease, and Renee and Meyer Luskin Family Fund.
  We thank Rosaline Young, Mark Dodson, Hansong Deng, and Jina Yun for developing and optimizing the protocol (Clark et al., 2006; Deng et al., 2008; Yun et al., 2014).

Competing interests

We have no competing interests.

References

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  9. Kandul, N. P., Zhang, T., Hay, B. A. and Guo, M. (2016). Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila. Nat Commun 7: 13100.
  10. Kim, N. C., Tresse, E., Kolaitis, R. M., Molliex, A., Thomas, R. E., Alami, N. H., Wang, B., Joshi, A., Smith, R. B., Ritson, G. P., Winborn, B. J., Moore, J., Lee, J. Y., Yao, T. P., Pallanck, L., Kundu, M. and Taylor, J. P. (2013). VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations. Neuron 78(1): 65-80.
  11. Kimonis, V. E., Fulchiero, E., Vesa, J. and Watts, G. (2008a). VCP disease associated with myopathy, Paget disease of bone and frontotemporal dementia: review of a unique disorder. Biochim Biophys Acta 1782(12): 744-748. 
  12. Kimonis, V. E., Mehta, S. G., Fulchiero, E. C., Thomasova, D., Pasquali, M., Boycott, K., Neilan, E. G., Kartashov, A., Forman, M. S., Tucker, S., Kimonis, K., Mumm, S., Whyte, M. P., Smith, C. D. and Watts, G. D. (2008b). Clinical studies in familial VCP myopathy associated with Paget disease of bone and frontotemporal dementia. Am J Med Genet. Part A 146A (6): 745-757.
  13. Kimura, Y., Fukushi, J., Hori, S., Matsuda, N., Okatsu, K., Kakiyama, Y., Kawawaki, J., Kakizuka, A. and Tanaka, K. (2013). Different dynamic movements of wild-type and pathogenic VCPs and their cofactors to damaged mitochondria in a Parkin-mediated mitochondrial quality control system. Genes Cells 18(12): 1131-1143.
  14. Ma, P., Yun, J., Deng, H. and Guo, M. (2018). Atg1 mediated autophagy suppresses tissue degeneration in pink1/parkin mutants by promoting mitochondrial fission in Drosophila. Mol Biol Cell: mbcE18040243.
  15. Meyer, H., Bug, M. and Bremer, S. (2012). Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat Cell Biol 14(2): 117-123. 
  16. Ritson, G. P., Custer, S. K., Freibaum, B. D., Guinto, J. B., Geffel, D., Moore, J., Tang, W., Winton, M. J., Neumann, M., Trojanowski, J. Q., Lee, V. M., Forman, M. S. and Taylor, J. P. (2010). TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97. J Neurosci 30(22): 7729-7739.
  17. Ritz, D., Vuk, M., Kirchner, P., Bug, M., Schutz, S., Hayer, A., Bremer, S., Lusk, C., Baloh, R. H., Lee, H., Glatter, T., Gstaiger, M., Aebersold, R., Weihl, C. C. and Meyer, H. (2011). Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations. Nat Cell Biol 13(9): 1116-1123.
  18. Watts, G. D., Wymer, J., Kovach, M. J., Mehta, S. G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M. P. and Kimonis, V. E. (2004). Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 36(4): 377-381.
  19. Weihl, C. C., Pestronk, A. and Kimonis, V. E. (2009). Valosin-containing protein disease: inclusion body myopathy with Paget's disease of the bone and fronto-temporal dementia. Neuromuscul Disord 19(5): 308-315.
  20. Yun, J., Cao, J. H., Dodson, M. W., Clark, I. E., Kapahi, P., Chowdhury, R. B. and Guo, M. (2008). Loss-of-function analysis suggests that Omi/HtrA2 is not an essential component of the PINK1/PARKIN pathway in vivo. J Neurosci 28(53): 14500-14510.
  21. Yun, J., Puri, R., Yang, H., Lizzio, M. A., Wu, C., Sheng, Z. H. and Guo, M. (2014). MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin. Elife 3: e01958. 
  22. Zhang, T., Mishra, P., Hay, B. A., Chan, D. and Guo, M. (2017). Valosin-containing protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants. Elife 6: e17834.

简介

[ 摘要] p97 / cdc48 / Valosin 含蛋白(VCP)的错义突变导致包涵体肌病,额颞叶痴呆的Paget病(IBMPFD)和其他神经退行性疾病。IBMPFD的病理机制尚不清楚,也没有治疗方法。我们生成了在成人体内飞行肌肉中IBMPFD的果蝇模型。在这里,我们描述了各种测定方法,以表征疾病病理和解剖疾病机制,以及体内VCP抑制剂的喂养后果。

[ 背景] VCP / p97 突变导致包涵体肌病,骨骼的Paget病和额颞叶性痴呆(IBMPFD),这是以常染色体显性方式在包括脑,肌肉和骨骼在内的多个系统中退化的疾病(Watts 等人,2004年) )。VCP的突变还与1-2%的散发性肌萎缩性侧索硬化症(ALS)以及遗传性痉挛性截瘫和Charcot-Marie-Tooth 2神经病有关(Abramzon 等,2012; de Bot 等,2012; Gonzalez 等,2014)。R155H突变是患者中最常见的突变,而具有A232E突变的个体具有最严重的临床表现(Kimonis 等,2008a; Ritson 等,2010)。90%的IBMPFD患者出现肌病,这是最早的症状(Weihl 等,2009)。50%的患者会发展成佩吉特氏骨病,影响头骨,脊柱,臀部和长骨。三分之一的患者发生额颞叶痴呆(Kimonis 等,2008b; Weihl 等,2009)。患者最终发展为心肺衰竭(Kimonis 等,2008b; Weihl 等,2009)。VCP 编码高度保守且丰富的AAA + ATP酶,其参与多个细胞过程(Meyer 等,2012)。由于VCP组装成六聚体,因此具有增强的ATPase活性的疾病突变体是通过显性激活机制(Chang 等,2011)还是显性阴性机制(Ju 等,2009; Ritz 等,2009 )引起争议。,2011; Bartolome 等,2013; Kim 等,2013; Kimura 等,2013)。我们建立了体内IBMPFD模型,以了解该疾病的发病机理并找到潜在的治疗方法。

对于IBMPFD疾病苍蝇的具体检测包括原位细胞凋亡检测,肌肉蛋白印迹试验,疾病标志物免疫荧光染色,肌肉的完整性检测/甲苯胺蓝染色和肌肉线粒体超微结构研究/电子显微镜(EM)成像,并在体内VCP抑制剂的治疗(Yun 等人,2008; Zhang 等人,2017; Ma 等人,2018)。

关键字:果蝇, 包涵体肌病, 佩吉特病和额颞痴呆(IBMPFD), VCP/p97, 线粒体, 肌肉, 疾病模型, 抑制剂, 药物治疗

材料和试剂


 


剃须刀片(VWR北美,目录号:55411-050 )
微量离心管(Denville Scientific Inc.,目录号:C2171)
PCR管(美国科学)
100平方目铜网上的FORMVAR FILM(电子显微镜科学,目录号:FF100-Cu)
在2 x 1 mm椭圆形槽铜网格上的FORMVAR碳膜(电子显微镜科学,目录号:FCF2010-Cu)
一次性药丸杵(Kimble,目录号:749521-0500)
PCR管
解剖盘
针头
移液器技巧
Toothpic K(飞世尔科技教育,S04180 )
0.22 µm过滤器(Millipore 和MillexGP 过滤器)
铝箔(Fisher Scientific ,目录号:01-213-101)
Immobilon-P PVDF转移膜(Millipore,目录号:IPVH00010)
果蝇菌株:UASt -VCP WT,VCP RH和AE系是慈康生博士的礼物(Chang 等,2011)。
果蝇菌株
IFM-Gal4,其中Gal4在间接飞行肌肉启动子的控制下,源于flightin 基因(Yun 等人,2014)。的IFM 启动子提供表达的强烈的脉冲在后期蛹期和成年期的最初几天后,被沉默(Kandul 等人。,2016) 。果蝇菌株保持在25°C的潮湿培养箱中。


20%多聚甲醛,EM级(电子显微镜科学,目录号:15713S)
8%戊二醛溶液,EM级(电子显微镜科学,目录号:16020)
16%多聚甲醛水溶液,EM级(电子显微镜科学,目录号:15710)
施耐德缓冲器(GIBCO,货号:21720-024)
原位细胞死亡检测试剂盒(Roche,目录号:11684795910)
卢damine毒伞素(生命科技,目录号:R415)
牛血清白蛋白(Fisher生物试剂,目录号:BP1600-100)
Fluoromou Ñ 吨-G(南方生物技术公司,目录号:0100-01)
Pierce RIPA 裂解液(Thermo Scientific,目录号:89900)
不含EDTA的Pierce蛋白酶抑制剂片剂(Thermo Scientific,目录号:88266)
Laemmli SDS样品缓冲液,6x(Bioland Scientific LLC,目录号:SAB02-02)
抗小鼠IgG辣根过氧化物酶连接的完整抗体(来自绵羊)(GE Healthcare,目录号:NXA931)
抗兔IgG辣根过氧化物酶连接的F(ab')2片段(来自驴子)(GE Healthcare目录号:NA9340V)
山羊抗兔/小鼠Alexa Fluor 546/488二抗(Life Technologies,目录号:A11034 / A11029 / A11035 / A10036)
吐温20 500毫升(Hoefer ,目录号:56-40-6)
Immobilon Western化学发光HRP底物(密理博,目录号:WBKLS0500)。
Embed812(电子显微镜科学,目录号:14900)
四氧化三((OsO 4 )4%水溶液(电子显微镜科学,目录号:19150)
DDSA(十二碳烯基琥珀酸酐)(电子显微镜科学,目录号:13710)
NMA(无水萘甲酸甲酯)(电子显微镜科学,目录号:19000)
BDMA(苄基二甲基胺)(电子显微镜科学,目录号:11400)
环氧丙烷(电子显微镜科学,目录号:20401)
甲苯胺蓝O粉末(电子显微镜科学,目录号:22050)
硼酸钠(Fisher Scientific,目录号:S249-500)
醋酸铀酰二水合物(Ted Pella,目录号:19481)
硝酸铅(Sigma-Aldrich,目录号:22862-100G)
二水合柠檬酸钠(EMD,目录号:SX0442-1)
氢氧化钠认证的ACS微丸NaOH(Fisher Scientific,目录号:S318-500)
NMS-873(3- [3-(环戊硫基)-5-[[[2-甲基-4'-(甲磺酰基)[1,1'-联苯基] -4-基]氧基]甲基] -4H-1, 2,4-三唑-4-基]-吡啶(Selleckchem ,目录号:S7285)
ML240,2- (2-氨基-1H-苯并咪唑-1-基)-8-甲氧基-N-(苯甲基)-4-喹唑啉胺(Sigma-Aldrich,目录号:1346527-98-7)
Permount 封固剂(Fisher Scientific公司,目录号:SP15-100)
0 .1%特里顿X - 100
PBS
TUNEL酶
β- 巯基乙醇
乙醇
肌肉解剖固定缓冲液(请参见食谱)
解剖缓冲区(请参见食谱)
TUNEL阻塞缓冲区(请参阅食谱)
0.2M磷酸盐缓冲液(p ħ = 7.4)(见配方)
10 ml EM固定液(请参阅食谱)
Epon Mix(中等)(请参阅食谱)
1%甲苯胺蓝染色溶液(请参见食谱)
醋酸铀染色溶液(请参阅食谱)
柠檬酸铅染色液(请参见配方)
 


设备


 


移液器(Denville Scien tific Inc. ,型号:P10,P 20,P 200,P 1000)
Ultra 45°金刚石刀(硅藻,目录号MX5341)
PELCO反向自闭合Twee zers(Ted Pella,目录号:5373 / 5375-NM)
DUMONT生物级镊子(Ted Pella,目录号:505 / 505-U)
粒料杵马达(金布尔/ Kontes ,目录号:749540-0000)
JEOL 100CX透射电子显微镜(加州大学洛杉矶分校脑研究所,电子显微镜核心设施)
超切超薄切片机(Leica EM UC6,Frank Laski博士,UCLA分子细胞与发育生物学)
PELCO Pro反向(自动关闭)t 镊子(Ted Pella,目录号:5375-NM)
解剖镊子(瑞士杜蒙生物学,电子显微科学)
金刚石刀(DiATOME ,Ultra 45°MX5341)
Formvar网格/槽网格(电子显微科学,目录号:FCF2010-Cu,FF100-Cu-50)
水浴(Fisher Scientific ,型号:Isotemp )
迷你PROTEAN ® 四手工浇铸系统和四印迹bodule (Bio-Rad公司)
安全通风柜(UCLA设施4315-3309-2)
加热块(Fisher Scientific,型号:Isotemp )
 


程序


 


间接飞行肌肉解剖
麻醉剂在CO 2 板上飞行。用剃须刀割断头部和腹部。
室温下,在微量离心管或PCR管中,在4%多聚甲醛/施耐德缓冲液中固定胸腔45分钟。
将固定后的胸带转移到含有0.1%Triton X -100 / PBS 的解剖盘中。Triton X - 100有助于打破表皮的表面张力,并在以下步骤中促进肌肉块的解剖。
间接飞行的肌肉(IFM,在图1A和1B中用红色箭头表示)位于胸骨的中间。可以使用30号针头或镊子解剖IFM。用针头或镊子轻轻地从背侧或腹侧中线打开胸腔,并暴露IFM。IFM是在胸中部前后左右运行的两大块肌肉(图1C)。每组IFM包含5条大块肌肉,这些大块肌肉彼此平行,并位于半剖开的胸部的背面。通过在将肌肉附着到角质层的末端进行切割来仔细隔离每条肌肉。一个固定良好的野生型胸部可以产生5-10个完整的肌肉碎片(图1C)。携带带有IFM靶向GFP标记的果蝇可用于进一步确认已经解剖了正确的肌肉(图1C)。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-5-20 \ 0101540--1347 MingGuo 495304 \ Figs jpg \图1-updated.jpg


图1 。间接飞行肌(IFM)解剖。A. IFM在完整胸部中的位置(红色箭头)。B.固定胸部的矢状面,表示红色箭头其中IFM 是LO cated。C.在解剖盘中解剖肌肉块。所用果蝇的基因型是IFM-Gal4> UASt-mitoGFP 。比例尺:0.5 毫米。


 


IBMPFD果蝇的原位细胞死亡检测/ TUNEL分析
固定胸骨并按上述方法从WT,VCP WT,RH和AE蝇中分离出IFM ,并收集到PCR管中。
将TUNEL封闭缓冲液引入含有肌肉的试管中30分钟。TUNEL封闭缓冲液含有0.2%的Triton X - 100,可同时渗透和封闭肌肉样品。
从原位细胞死亡检测试剂盒中加入TUNEL酶(4 µl)和10x反应缓冲液(36 µl),并在37 °C的水浴中温育2-3小时。每隔30分钟使用移液器轻轻地混合样品。
用PBS洗涤样品两次。
用Fluoromount -G安装溶液替换PBS 。上使用一个玻璃吸管或200显微镜载玻片安装的IFM μ 升移液管尖端,并用共聚焦显微镜可视化的肌肉使用的546纳米的激发波长。在6天大的果蝇中,VCP RH和AE表达导致明显的细胞死亡(Zhang 等人,2017)。观察到这是广泛的红色核染色。
 


免疫荧光染色和共聚焦显微镜成像
如上所述,解剖固定的肌肉。然后用PBS洗涤,在室温下在摇杆上用0.2%Trito n X - 100 / PBS 渗透肌肉3-4小时,然后用1 x PBS 洗涤5 分钟。
对于肌原纤维染色,添加若丹明鬼笔环肽(1:500稀释)并在室温或4°C下染色2 小时过夜。
为了进行免疫荧光染色,将固定的肌肉片段与在0.2%Triton X - 100 / PBS中稀释至所需浓度(1:100或1:200)的一抗在4°C 孵育过夜。我们使用浓度为1:100的抗TARTAR结合蛋白或抗TDP43。
用0.2%Triton X - 100 / PBS 洗涤3次,每次10分钟。
将肌肉片段与山羊抗兔/小鼠Alexa Fluor 488或546二抗(1:200在0.2%Triton X - 100 / PBS中)在室温或4°C下孵育过夜。
用0.2%Triton X - 100 / PBS 洗涤3次(每次10 分钟),并将样品置于带有Fluoromount -G 的显微镜载玻片上。在共聚焦显微镜下以对应于第二抗体的在488nm或546nm的激发波长使样品可视化。在6天大的果蝇中,TDP43定位于野生型果蝇的IFM的细胞核和胞浆/胞质区域。在VCP RH和AE突变体的肌浆/胞质区域中,细胞核的定位模式消失了,并观察到更多的聚集体(Zhang et al 。,2017)。
 


蛋白质裂解物和蛋白质印迹
用CO 2 麻醉苍蝇,如上所述分离出胸部,然后将它们放入冰上的微量离心管中。
注意:胸腔未固定。


加入带有蛋白酶抑制剂的RIPA裂解缓冲液,每个胸腔10-15 µl,每个基因型5-10胸腔。
将裂解颗粒中的胸部均质,然后将聚丙烯颗粒杵预冷5-6次,然后用研钵以最高速度旋转杵10-15 s。在冰上孵育裂解物30分钟以完全溶解组织。
将蛋白质以10,000 xg的速度离心15分钟,然后将上清液转移至干净的微量离心管中。
在95 °C下用6x SDS样品缓冲液和β- 巯基乙醇煮沸上清液5分钟。
加载蛋白质,让它们在Tris / SDS运行缓冲液中的8%SDS-PAGE凝胶中分离,并在80伏特下运行。
转移蛋白至PVDF膜,用于在Tris /甘氨酸缓冲液,用15%2小时米乙醇。
用3%BSA / PBS孵育转移膜1小时。
将膜与稀释在1%BSA / 0.01%Tween-PBS中的一抗在4 °C下孵育过夜。
将膜在0.01%Tween-PBS中洗涤3次(每次10分钟),并与在5%脱脂牛奶/0.01% Tween-PBS中稀释的抗小鼠/兔IgG HRP连接的二抗孵育2小时。
用Immobilon Western化学发光HRP底物试剂盒开发膜。我们在胸腔中以可比较的水平表达了VCP WT,VCP RH和AE (Zhang et al 。,2017)。
 


IBMPFD果蝇的甲苯胺蓝染色和电子显微镜
固定和嵌入


注意:在安全通风橱中执行步骤E 4和后续步骤。


麻醉苍蝇并切开胸骨,迅速浸入95%乙醇中(这有助于破坏表面张力,以便于随后将其浸入EM固定液中)。然后将胸液转移至微量离心管中的0.5 ml冰老EM固定缓冲液(下)中。
定出一个在冰上至少2小时以摇动。如果固定时间较长或需要暂停操作,则可在2 h时间点后将样品留在固定剂中,并在4 °C下放置一整夜或直到准备进行。荆棘将沉入微量离心管的底部。
在室温下,用0.1 M磷酸盐缓冲液冲洗每个样品3次(每次10分钟)。
注意:所有后续步骤均应在生物安全罩中进行。


将样品在ddH 2 O 中的1%OsO 4 (新鲜制备)中于室温下固定2 h。样品用OsO 4 固定的时间不得超过2小时,否则样品会变脆。
在室温下用ddH 2 O 冲洗样品3次(每次10分钟)。
在室温下将样品分别在70%和95%乙醇中脱水5分钟。
在室温下,将样品在100%乙醇中脱水两次(每次10分钟)。
在室温下,将样品在100%环氧丙烷中脱水两次(每次7分钟)。
在室温下将样品浸入100%环氧丙烷和Epon 混合物的1:1混合物中30分钟。
在室温下,将样品在100%Epon Mix(添加1.5%BDMA)中孵育过夜。
将样品以100%Epon 混合液(添加1.5%BDMA)引入包埋模具中。在每个孔的右下角,放一张带有样品名称/编号的纸(图2A)。
在6 0 -70 °C 过夜聚合Epon 。
C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-5-20 \ 0101540--1347 MingGuo 495304 \ Figs jpg \图2-updated.jpg


图2. 果蝇胸腔包埋和切片位置。答:两个单独的样本嵌入并用标签封闭。B. 正确嵌入˚F LY胸部(红色箭头)在的Epon 树脂。胸部的矢状平面(白色虚线)应平行于树脂块的边缘,以便于剖切。C. 修整好的树脂块,厚切片后有胸部样品。玻璃刀或金刚石刀的刀片应该与胸部的矢状面平行。比例尺:1 毫米。


 


切片和染色


甲苯胺蓝染色
如图所示,将嵌入良好的胸腔样本放置在树脂块中(图2B)。胸廓的矢状中线平行于树脂块的边缘。
使用剃刀刀片切掉的Epon 围绕SAMPL ë尽可能(图2C) 。减小截面面积将提高截面质量。
切割厚的部分(1.5-2 µm)以进行甲苯胺蓝染色,并确定将在其中进行电子显微镜成像的薄部分的区域。
用切片刀和玻璃刀切出较厚的部分。使用牙签将切片转移至载玻片上的水滴。
将载玻片放在70 °C的加热块上以蒸发水。这将使截面样本变平。
应用1%甲苯胺蓝以完全覆盖该切片样品区域,并将载玻片放在70 °C 加热块上,直到甲苯胺蓝液体的边缘开始干燥(2-5分钟)。不要过度干燥甲苯胺蓝溶液,否则切片会被染色缓冲液中的干燥溶质污染,从而导致图像质量下降。
在ddH 2 O中冲洗载玻片,直到去除未结合的污渍。
在使用Permount 安装溶液之前,先将部分完全干燥。
图3A中显示了野生型胸部对齐良好的厚切片样品(可以轻松观察到间接飞行的肌肉碎片)。可以利用组织样品的更高放大倍率图像(图3B)来分析肌肉组织的完整性。在6天大的果蝇中,与WT和VCP WT对照相比,表达VCP RH和AE的果蝇破坏了肌肉组织的完整性(Zhang et al 。,2017 )。
 


D:\ Reformatting \ 2020-3-2 \ 0101540--1347 MingGuo 495304 \ Figs jpg \图3.jpg


图3 。IFM的甲苯胺蓝和EM图像。AB。在不同放大倍数下,胸部厚部分的甲苯胺蓝染色。C. 野生型胸部薄片的EM图像。D.使用ImageJ对线粒体大小进行量化。小号Cale的棒:1 微米(119个像素)。选择的线粒体横截面面积为2.92 微米2 (红色小号quare)。


 


电子显微镜样品制备
从甲苯胺蓝染色中选择对齐良好的样品,以用于薄切片(80-90 nm)。
用钻石切片机(已消毒的ddH 2 O)用切片机切成薄片。这些部分应显示银色到金色。
用细针将1-4个部分放在水面上的一条线上以进行网格安装。
拿起一个没有用镊子涂上formvar的空插槽网格。轻轻触摸网格以使浮在表面上的部分变小。这些部分将连接到水滴内插槽的中间。
使用一对自闭合镊子,将新的密封密封的插槽网格或网状网格朝上放置。将附有分区的插槽网格放置到新formvar网格的光亮面上。
将三角形滤纸的尖角放在网格之间的边缘,以除去两个网格之间的水滴。这些部分将附加到formvar密封的新网格上并展平。
删除空的网格,然后重新使用它来拾取其他部分。的部分连接的网格可以是商店d 在进一步染色的网格框如下面描述的。
制备醋酸铀(UA)溶液,并通过0.22 µm过滤系统过滤。在室温下,在UA解决方案上将网格的截面面漂浮15分钟。在ddH 2 O水滴中清洗网格五次(每次1分钟),或在3个装有ddH 2 O的单独烧杯中清洗三遍(每次1分钟)。
将网格部分的光泽面朝下放在一滴柠檬酸铅溶液上,并在室温下染色5-10分钟。戴上口罩以防止呼吸,因为过量的CO 2 会影响染色质量。保持染色皿用染色皿随附的塑料盖覆盖。
按上述方法用ddH 2 O 洗涤格栅。
将染色的网格转移到网格盒中以进行进一步成像。
图3C显示了使用TEM以10,000x观察的野生型肌肉的图像。与野生型和VCP WT苍蝇相比,在6天大的苍蝇中,表达VCP RH和AE的苍蝇显示肌动蛋白和线粒体结构受到破坏(Zhang 等,2017 )。
线粒体横截面尺寸定量
一个。导入的超微结构Ë lectron显微镜图像(10,000 X MAG nification)在以图像J软件(美国国立卫生研究院),图3D。       


b。设置比例后(分析>设置比例:119像素= 1 µm),使用多边形选择(黄线)绘制图像上的每个线粒体并测量其面积(分析>测量),如图3D所示。图像上的所有线粒体均被单独测量。      


C。对于每个胸部分析至少三张图像,并检查每种基因型的3个胸部。       


d。一个独立的小号tudent的牛逼- 测试我š用来测试不同基因型之间的统计学意义。      


 


体内VCP抑制剂治疗
将NMS-873(19.2 mM,原料浓度)和ML240(25.2 mM,原料浓度)的粉末形式溶解在DMSO中作为原料。
稀储备溶液在等hanol /的DDH 2 O(100微升乙醇+ 600微升的DDH 2 O),以该所希望的浓度。乙醇可以帮助保持溶液中的化合物。在微波炉中加热果蝇食物20秒钟,直到其完全融化,然后使其冷却至<50°C。将食品中的DMSO /乙醇H 2 O中的化学物质与每4 毫升飞行食品中10升的食用染料一起添加到食物中,并手动混合直至颜色均匀。如果食物看起来很潮湿,可以在室温下将带有棉塞的小瓶干燥。相当数量的DMSO /乙醇用作载体对照。
将所需基因型的果蝇父母放在含有DMSO /乙醇或抑制剂的食物中3天,以产卵,然后取出。后代的生长则发生在存在的车辆控制或测试化合物。
之后立马 隔离成虫(从其case壳孵出),将这些成虫转移到新鲜制备的含有相同浓度DMSO或抑制剂的食物中,并在进行分析之前将果蝇喂养所需的天数。
VCP抑制剂喂养significantl ý反向ð 第ë肌肉崩解,肌肉细胞死亡和超微结构线粒体缺陷VCP RH和AE蝇(张等人。,2017) 。
 


菜谱


 


肌肉解剖固定缓冲液(500 µl)
100 µl 20%多聚甲醛


施耐德缓冲液400 µl


储存在4°C


注:镨eferably准备新鲜的每次实验。


解剖缓冲区
0.1%Triton X - 100


1x PBS缓冲液


室温保存


TUNEL阻塞缓冲区
的50mM Tris-Cl(上p ħ = 7.4)


188毫米氯化钠


Triton X-100的0.2%


1%BSA


储存在4°C


0.2M磷酸盐缓冲液(p ħ = 7.4)
溶液X:3.516 g Na 2 HPO 4· 2H 2 O / 100 ml ddH 2 O


溶液Y:2.76 g NaH 2 PO 4 · H 2 O / 100 ml ddH 2 O


将40.5 ml溶液X 与9.5 ml溶液Y混合,然后获得50 ml的0.2 M磷酸盐缓冲液。所述p ħ应该是7.4 。


室温保存


10 ml EM固定液
1.25毫升8%戊二醛


0.625毫升16%低聚甲醛


5.0 ml 0.2 M磷酸盐缓冲液


3.125 ml的的DDH 2 ö与不孕ë


储存在4°C


Epon Mix(中)
Embed812 20毫升             


DDSA(十二碳烯基琥珀酸酐)16毫升             


NMA(无水纳迪克甲基)8毫升             


BDMA(苄基二甲基胺)0. 9 毫升(每次嵌入均需新鲜添加)             


包埋前彻底混合至少1 小时


可以在室温下保存在避光容器中。预ferably每次准备新鲜


1%甲苯胺蓝染色液
1克甲苯胺蓝粉


1克硼酸钠


100毫升ddH 2 O


使用前过滤0.22 µm。


室温保存在避光容器中


醋酸铀染色液
4克乙酸铀酰


100毫升ddH 2 O


完全溶解(加热至70°C以促进溶解)


使用前过滤0.22 µm


室温下保存在防光照的丙烯酸储藏容器中,以防止β 辐射


铅CITR 吃染色液
在装有铝箔的小烧杯中煮沸100 ml ddH 2 O,以除去CO 2 ,使其冷却至室温


1.33克硝酸铅


1.76克柠檬酸钠


30 ml无CO 2 ddH 2 O


8毫升1 N NaOH(100毫升ddH 2 O中的4克NaOH )


添加无CO 2的ddH 2 O至总体积为50 ml


储存在4 °C的避光容器中


 


确认小号


 


我们感谢美国国立卫生研究院(国立衰老研究所),格伦医学研究基金会,娜塔莉·R和尤金·琼斯衰老与神经退行性疾病研究基金会,肯尼斯·格伦家庭基金会的慷慨支持。加州大学洛杉矶分校Laurie和Steven Gordon对帕金森氏症的治疗承诺,以及Renee和Meyer Luskin家庭基金会。


  我们感谢Rosaline Young,Mark Dodson,Hansong Deng和Jina Yun制定并优化了协议(Clark 等,2006; Deng 等,2008; Yun 等,2014)。


 


竞争我nterests


 


我们没有利益冲突。


 


 


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, T., Hay, B. A. and Guo, M. (2020). Generation, Analyzing and in-vivo Drug Treatment of Drosophila Models with IBMPFD. Bio-protocol 10(10): e3621. DOI: 10.21769/BioProtoc.3621.
  2. Zhang, T., Mishra, P., Hay, B. A., Chan, D. and Guo, M. (2017). Valosin-containing protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants. Elife 6: e17834.
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