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Mar 2020
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Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans
利用光遗传学淀粉样蛋白-β监测黑腹果蝇、斑马鱼和秀丽隐杆线虫体内蛋白质聚集   

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Abstract

Alzheimer’s Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in three model organisms–D. melanogaster, C. elegans and D. rerio. We generated a fluorescently labeled, optogenetic Aβ peptide that oligomerizes rapidly in vivo in the presence of blue light in all organisms. Here, we detail the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and performing metabolic assays to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.

Keywords: Optogenetics (光遗传学), Amyloid-β (淀粉样蛋白-β), Alzheimer’s Disease (阿尔兹海默症), Light-sheet (光片), Drosophila melanogaster (黑腹果蝇), Zebrafish (斑马鱼), Caenorhabditis elegans (秀丽隐杆线虫)

Background

Alzheimer’s disease (AD) is a debilitating, age-associated, neurodegenerative disease (Zhang et al., 2011; De-Paula et al., 2012; Kumar et al., 2015). The accumulation of amyloid beta (Aβ) as extracellular Aβ plaques is believed to be a major cause of the disease (Hardy and Higgins, 1992), but the failure of interventions that target these Aβ plaques and their presence in brains of non-AD symptomatic individuals (Hawkes, 2016; Cummings et al., 2018) suggest further mechanistic analysis is required. An alternative hypothesis of neurotoxicity induced by soluble intracellular Aβ aggregates has been proposed (LaFerla et al., 2007; Ferreira and Klein, 2011), but currently there is a dearth of tools to control Aβ oligomerization in vivo to study the effects of soluble Aβ aggregates on disease progression and the therapeutic potential of drugs.

Optogenetics is the ideal tool for this purpose as it allows highly precise spatial and temporal regulation of proteins in vivo simply by exposure of the organism to light of a specific wavelength (Moglich and Moffat, 2010; Fenno et al., 2011). We have applied this approach to a variety of problems including embryonic development and homeostasis (Kaur et al., 2017; Bunnag et al., 2020), and have recently shown that inducing aggregation of soluble Aβ protein using optogenetics led to metabolic and physical damage and deteriorating lifespan and healthspan in 3 model organisms–Drosophila melanogaster, C. elegans and D. rerio, as well as in standard tissue culture models (Lim and Mathuru, 2017; Lim et al., 2020). Here, we describe the method used to construct an optogenetic light-inducible Aβ protein that oligomerizes on exposure to blue light in vivo in different model organisms and investigate the downstream detrimental effects using various metabolic assays and time-lapse live imaging. We used the photolyase homology region of the Arabidopsis thaliana cryptochrome 2 as an optogenetic switch (CRY2) protein that oligomerizes into photobodies quickly and reversibly in the presence of blue light (Mas et al., 2000; Bugaj et al., 2013). This CRY2 domain was generated as a Aβ-CRY2-mCherry fusion protein to induce aggregation of Aβ protein upon activation by blue light with visualization through the mCherry fluorescent protein.

Incorporation of this versatile tool with easily manipulatable genetic models such as D. melanogaster, C. elegans and D. rerio, gave us temporal control over the induction of soluble Aβ oligomers not feasible with other currently available Alzheimer’s animal models. We were also able to dissect the pathological effects of Aβ oligomers on embryos during development in real-time using live imaging as well as in adult flies (Lim et al., 2020). This model also allows us to generate mosaics of embryos such that only one half of the embryo, or a small portion is exposed to blue light. Hence, we can investigate the effects of targeted light-induced Aβ oligomerization on various anatomical structures such as the nervous system with the unlit portion of the embryo acting as the control. Functionality in both the invertebrate and vertebrate lab models demonstrates this technique’s application to study the biochemistry, metabolism, cellular and neuronal biology of induced Aβ oligomerization. The effects of potential drug interventions on lifespan, health span and metabolism can also be studied with this robust optogenetic approach in various model organisms. We were able to adopt different strategies to express the same transgene in both invertebrates and vertebrates, which should translate to mouse studies through the conditional knock-in approach. Aggregation could be induced in brains of adult mice with exposure to blue light to study the effects of Aβ-CRY2-mCh aggregation on neuronal function and behavior. This protocol is specific to Aβ, but could easily be adapted to other aggregating proteins simply by changing the starting gene.

Materials and Reagents

  1. Molecular cloning of optogenetic transgenes

    1. 10 cm Petri dishes (Greiner Bio One International, catalog number: 633185)

    2. 14 ml tubes PP (Bio Lab, catalog number: 352059)

    3. 1.5 ml Micro tubes (Axygen, catalog number: MCT-150-L-C)

    4. Pipette tips (fitting for 100-1,000 μl, 20-200 μl, 2-50 μl and 0.5-10 μl pipettes)

    5. Microloader (Eppendorf)

    6. pUCIDT-attL1-Human ABeta-attR5 (Addgene: 160436)

      Note: The Aβ human gene was synthesized with attL1 and attR5 overhangs into a Kanamycin resistant plasmid backbone for use as a Gateway entry vector.

    7. pUCIDT-attL1-Worm ABeta-attR5 (Addgene: 160437)

      Note: The Aβ worm gene was synthesized with attL1 and attR5 overhangs into a Kanamycin resistant plasmid backbone for use as a Gateway entry vector.

    8. pDONR-attL5-CRY2-mCh-attL2 (Addgene: 160438)

      Note: CRY2-mCh was amplified from pCRY2PHR-mCherryN1 (which was a gift from Chandra Tucker (Addgene plasmid, catalog number: 26866) (Kennedy et al., 2010). attB5 and attB2 sites were added to the CRY2-mCh PCR product and recombined into pDONR P5-P2 vector. The final pDONR-attL5-CRY2-mCh-attL2 plasmid was used as a Gateway entry vector.

    9. pUASg-HA-attB (Drosophila Genomics Resource Center, stock number: 1423)

      Note: Vector specific sequencing primers used were HSP

      Forward: TATAAATAGAGGCGCTTCGT

      HA Reverse: AGCGTAATCTGGAACGTCATA

    10. pDEST-hsp16-2p (a kind gift from Hidehito Kuroyanagi, Medical Research Institute, Tokyo Medical and Dental University)

      Note: Vector specific sequencing primer used was Hsp16-2p: CGAATGTGAGTCGCCCTCCT

    11. pDEST-Tol2-PA2-CMV (Tol2 kit). Used in molecular cloning of following transgenes for Danio rerio – a) pDEST-Tol2-PA2-10xUAS-Aβ-CRY2-mCh; b) pDEST-Tol2-PA2-Ubi-Aβ-CRY2-mCh; c) pDEST-Tol2-PA2-CMV-Aβ-mCh (Addgene ID: 160435)

    12. Top 10 competent cells (Thermo Fisher Scientific, catalog number: C404003)

    13. Agarose, Biotechnology Grade, 500 g (1st Base, catalog number: BIO-1000-500g)

    14. LB Agar Miller, Bacterial Culture media, 500 g (1st Base, catalog number: BIO-4010-500g)

    15. LB Broth Miller, pH 7.0, Bacterial Culture media, 500 g (1st Base, catalog number: BIO-4000-500g)

    16. Ampicillin sodium salt (Sigma, catalog number: A9518G)

    17. Kanamycin sulfate (Sigma, catalog number: K1377)

    18. MultiSite Gateway® Pro Plus kit (catalog number: 12537100)

    19. GatewayTM LR ClonaseTM II Enzyme mix (Thermo Fisher Scientific, catalog number: 11791100)

    20. GatewayTM BP ClonaseTM Enzyme mix (Thermo Fisher Scientific, catalog number: 11789020)

    21. Phusion High-Fidelity PCR kit (Thermo Fisher Scientific, catalog number: F553S)

    22. Gibson Assembly® Cloning kit (New England Biolabs, catalog number: E5510S)

    23. Low melting point agarose (Promega, catalog number: V2111)


  2. Expression of optogenetic transgenes in neurons in vivo (for D. melanogaster)

    1. 1 L microwave-safe glass beaker

    2. Magnetic stirrer

    3. 50 ml glass beaker

    4. Spatula

    5. Compressed carbon dioxide gas cylinder (any brand)

    6. LED lamp (any brand)

    7. Aluminum foil (any brand)

    8. Styrofoam box (any brand)

    9. Fly cages for 60 mm Petri dishes (any, e.g., Small Embryo Collection Cage For 60 mm Petri Dishes, FlyStuff, catalog number: 59-100)

    10. 60 mm Petri dishes (any brand, e.g., NuncTM EasYDishTM Dishes, ThermoFisher Scientific, catalog number: 150462)

    11. Drosophila sorting brush (any brand) or a very thin paint brush

    12. Drosophila [Bloomington Drosophila Stock Center (NIH P40OD018537)]. Stock number 458, elav promoter driving GAL4 for the GAL4/UAS system (Brand and Perrimon, 1993)

    13. P(ET-QF2.GU)repo [Q system from Bloomington Drosophila Stock Center (NIH P40OD018537), stock number: 66477] (Lin and Potter, 2016)

    14. P(QUAS-mCD8-GFP)X (Q system from Bloomington Drosophila Stock Center [NIH P40OD018537), stock number: 30001) (Lin and Potter, 2016)

    15. Bacto agar

    16. Glucose

    17. Cornmeal

    18. Brewer’s yeast

    19. Nipagin

    20. FlyStuff Grape Agar Premix, For Embryo Collection (25 Packets/Unit) (FlyStuff, catalog number: GEN47-102)

    21. Yeast paste (Bruggeman Instant Dry Yeast Blue, 500g)

    22. Standard fly food (see Recipes)

    23. Grape juice agar plates (see Recipes)

    24. Yeast paste (see Recipes)


  3. Crosses and expression of constructs (for D. rerio)

    1. 3 ml Pasteur Pipette (Tarson, catalog number: T940050)

    2. Tg gng8:Gal4 (with thanks to Prof. Marnie Halpern) (Hong et al., 2013)

    3. Tg UAS:GCamp6S in nacre background (mitfa-/-)

    4. Tricaine (MS-222 or Ethyl 3-aminobenzoate methanesulfonate; Sigma, catalog number: A5040)

    5. NaCl

    6. KCl

    7. CaCl2·2H2O

    8. MgSO4·7H2O

    9. Tris-HCl

    10. 25x Tricaine stock solution (for D. rerio)

    11. 60x E3 stock solution (see Recipes)


  4. Microinjection and expression of UAS construct (for D. rerio)

    1. Standard wall borosilicate glass with filament (SUTTER INSTRUMENT, model: O.D 1 mm, I.D 0.5 mm, Length 7.5 cm, catalog number: BF100-5-7.5)


  5. Light-sheet microscopy to image neural development during embryogenesis (for D. melanogaster)

    1. Disposable dropper (any brand)

    2. 15 ml Falcon tubes (any brand)

    3. Stainless steel dissecting needle, L160 mm (Hammacher, HWO010-16, catalog number: 91-2483)

    4. Glass capillary, size 2 black, inner diameter of capillary ~1 mm (BRAND GmbH, catalog number: 701932) with corresponding plunger

    5. 5 cm long electrical copper wire (or any other fine tip wire)

    6. Weighing boat

    7. Halocarbon oil 27 (Chemical Raw Materials Ltd, catalog number: 3904 9000)

    8. Bleach (Clorox)

    9. Autoclaved MilliQ water

    10. 1% Agarose, low gelling temperature Type VII-A (Sigma, catalog number: A0701) dissolved in water aliquoted into 1.5 ml Eppendorf tubes (see Recipes)


  6. Light-sheet microscopy to image Aβ-CRY2-mCh in neurons in fish (in D. rerio)

    1. Heating block for mounting medium


  7. D. rerio Mitochondrial metabolic flux assay

    1. Oligomycin (Sigma-Aldrich, catalog number: 75351)

    2. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920)

    3. Sodium azide (Sigma-Aldrich, catalog number: C2002)

    4. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)


  8. D. rerio Adenosine triphosphate (ATP) assay

    1. Firefly Lantern Extract (Sigma-Aldrich, catalog number: FLE250)

    2. MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391)

    3. KH2PO4 (Sigma-Aldrich, catalog number: P0662)

    4. Na2HASO4·7H2O (Sigma-Aldrich, catalog number: S9663)

    5. TCA powder (Sigma-Aldrich, catalog number: T6399)

    6. 10% Trichloroacetic acid (TCA) solution (see Recipes)

    7. Arsenite ATP buffer (see Recipes)

    8. 25x Tricaine stock solution (for D. rerio) (see Recipes)


  9. Fluorescence microscopy to image Aβ aggregates in C. elegans

    1. 1.5 ml Eppendorf tube (any brand)

    2. Levamisole hydrochloride (Sigma, catalog number: 196142)

    3. 2% Agarose (Sigma, catalog number: A0701) dissolved in water aliquoted into 1.5 ml Eppendorf tubes (see Recipes)

    4. 2% low melting agarose (see Recipes)

Equipment

  1. Expression of optogenetic transgenes in neurons in vivo (for D. melanogaster)

    1. Drosophila workstation with CO2 pad (Flowbuddy Complete; Genesee Scientific, catalog number: 59-122WC)

    2. Incubator for 25 °C incubation of flies (any brand, e.g., INVICTUS Drosophila Incubator, Genesee Scientific)

    3. Stereo microscope (any brand, e.g., Leica, model: M50)

    4. Fluorescence stereo microscope (any brand, e.g., Leica MZ16F)


  2. Light-sheet microscopy to image neural development during embryogenesis (for D. melanogaster)

    1. Dissecting fine-pointed serrated forceps (any brand)

    2. Heat block with temperature range 37 °C and 70 °C (any brand, e.g., Digital block heater, Thermo Fisher Scientific)

    3. Zeiss Light-sheet Z.1 (Carl Zeiss)

    4. Sample chamber


  3. For experiments with D. rerio

    1. Incubator for bacterial culture, 37 °C

    2. Shaker incubator for bacterial culture, 37 °C

    3. Light-sheet microscopy to image Aβ aggregation in imaging zebrafish

    4. Seahorse extracellular flux analyzer (Agilent Technologies, Seahorse Bioscience, model: XFe96)

    5. Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Instruments)

    6. Micropestle homogenizer


  4. Fluorescence microscopy to image Aβ aggregates in C. elegans

    1. Incubator for zebrafish embryos, 28 °C

    2. Forceps (F.S.T, model: Student Fine Forceps, Straight, catalog number: 91115-10)

    3. Micropipette puller (SUTTER INSTRUMENT, model: P-1000)

    4. Microinjection system (Harvard Apparatus, model: PLI-90 Pico-injector)

    5. Confocal microscope (Zeiss, model: LSM800)


  5. Other assays used to confirm aggregation-induced deficits in C. elegans

    1. Incubator (Aqualytic, TC 255 S)

    2. Seahorse extracellular flux analyzer (Agilent Technologies, Seahorse Bioscience, model: XFe96)

    3. Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Instruments)

Software

Light-sheet microscopy to image neural development during embryogenesis

  1. ZEN 2014 Processing software package for Light-sheet Z.1 (Carl Zeiss, catalog number: 410136-1059-130)

  2. MetaMorph® Microscopy Automation and Image Analysis Software (Molecular Devices)

Procedure

  1. Molecular cloning of optogenetic transgenes for expression in D. melanogaster and C. elegans

    Use the human Aβ1-42 amino acid sequence (Aβ Human) to generate transgenes for Drosophila and zebrafish. For C. elegans, use a nematode-codon-optimized version of Aβ1-42 (Aβ Worm) (Fong et al., 2016). For Drosophila expression of human Aβ-CRY2-mCh, obtain the human Aβ DNA sequence with attL1 and attR5 overhangs in a Gateway entry vector (pUCIDT-attL1-Human ABeta-attR5) and the pDONR-attL5-CRY2-mCh-attL2. Perform a multi-site Gateway LR reaction to insert these constructs into the pUASg-HA-attB Drosophila expression vector (Bischof et al., 2007), according to the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific). For C. elegans expression of worm Aβ-CRY2-mCh, perform a multi-site Gateway LR reaction between the pUCIDT-attL1-Worm ABeta-attR5 entry vector, the pDONR-attL5-CRY2-mCh-attL2 and the pDEST-hsp-16–2p C. elegans expression vector, as per the manufacturer’s instructions (Invitrogen, ThermoFisher Scientific). For all transgenes generated, use vector specific primers for sequencing to confirm the orientation and insertion of both fragments into the destination vector.


  2. Molecular cloning of optogenetic transgenes for Danio rerio (zebrafish) – a) pDEST-Tol2-PA2-10xUAS-Aβ-CRY2-mCh; b) pDEST-Tol2-PA2-Ubi-Aβ-CRY2-mCh; c) pDEST-Tol2-PA2-CMV-Aβ-mCh

      Cloning single DNA element of interest into gateway compatible vector
    1. PCR amplify 10xUAS, Ubi and CMV sequence with specific primer flanked by attB4 (Forward primer) and attB1 (Reverse primer) and cloned into pDONRTMP4-P1R to generate 5' entry plasmid via BP recombination. Verify correct clones by sequencing primers M13F or 5' GTAAAACGACGGCCAGT3' and M13Rev 5' CAGGAAACAGCTATGAC3'.

    2. PCR amplify the Aβ-CRY2-mCh with specific primer flanked attB1 (Forward primer) and attB2 (Reverse primer) and cloned into pDONR221TM to generate middle entry plasmid via BP recombination. Verify correct clones by M13 primers (same as 1).

      Note: The 3' entry vector containing poly-A signal (p3' PolyA) and the Destination plasmid containing the Tol2 recognition sites (pDestTol2PA2) originated in the Kawakami Laboratory and are available as–Tol2 kit (Kawakami, 2007).

    3. Create compatible attL sites on each entry plasmids following BP recombination. LR recombination to generate expression plasmid. Verify correct clones by sequencing primers

      Forward: 5' CAAGTTTGTACAAAAAAGCAGGCTTA3'

      Reverse: 5' TACCCAGCTTTCTTGTACAAAGTGGGGGA 3'.

    4. Summary of the fragment assembly is shown in Figure 1.



      Figure 1. Generation of expression clones for zebrafish transgenic


  3. Expression of optogenetic transgenes in neurons in vivo

    For D. melanogaster, inject the pUASg-humanAβ-CRY2-mCh-HA-attB transgene into attP2 (Strain#8622) P[CaryP]attP268A4 (BestGene Inc (California)) (Groth et al., 2004; Markstein et al., 2008). Transgenic flies will be provided upon request. Isolate at least 5 homozygous male UAS-Aβ-CRY2-mCh flies and cross these with at least 10 Elav-Gal4, Repo-QF2; QUAS-GFP female virgin flies to drive neuronal expression of Aβ-CRY2-mCh and glial expression of GFP in the F1 generation. Place these male and female flies in a fly cage and cover with a pre-warmed grape juice agar plate smeared with a small amount of yeast paste to encourage egg laying. Keep the cages in a 25 °C incubator. For control flies, use UAS-TdTomato homozygous males instead. Change the fly plates every day and replace with pre-warmed grape juice agar plates that have a small amount of yeast paste on them. Allow newly set up cages to lay eggs for about 3 days to ensure a good number of developing embryos before using them for experiments.

      For C. elegans, obtain transgenic strain (gnaEx1[myo-2p::YFP+hsp-16-2p::Abeta1-42:: CRY2::mCherry]) from Caenorhabditis Genetics Center (CGC). Age-synchronize transgenic nematodes through hypochlorite bleaching (Stiernagle, 2006). To induce in vivo expression of Aβ-CRY2-mCh driven by the hsp16-2 promoter, incubate day 1 adult nematodes at 35 °C for 1 h. After 1 h incubation, separate the nematodes into light/dark groups and grow them in a 20 °C incubator. For dark treatment, wrap the plates containing nematodes with aluminum foil and place the wrapped plates inside a Styrofoam box. Place the Styrofoam box at the top of incubator rack to avoid exposure to the LED light. For light treatment, place the plates containing nematodes directly under the LED light on the bottom rack of the incubator.

      For D. rerio: Zebrafish lines (with Gal4; UAS) express the transgene in specific neurons or cells. To prevent oligomerization in ubiquitously expressing larvae, raise injected embryos in a dark incubator until experimentation. Minimize light exposure for transferring plates out or for sorting to avoid exposure to high LED light. For light treatment for mitochondrial flux analysis or ATP measurements, place the larvae under white LED light of a stereoscope for 2 h. Larvae with more restricted expression such as those used in light-sheet imaging were less sensitive to light exposure but were still raised in the dark till imaging as described in the following sections.


    Microinjection and expression of UAS construct (preparation on Day -1)

    1. The day before the injection, set up the fish in breeding tanks with dividers (Video 1).


      Video 1. Zebrafish microinjection


    2. Using a strainer, collect the embryos and rinse with tap water before transferring them into a 10 cm Petri dishes containing E3 (1x). Remove unfertilized eggs and debris with a Pasteur pipette.

    3. Prepare the injection needle by micropipette puller and backfilled with 3 μl of the mixture listed in the table (Table 1) using a Microloader.

    4. Fix the needle onto the micromanipulator connected to the microinjection system.

    5. Make a microinjection plate with 3% agarose using a custom-built plastic mold with grooves.


      Table 1.Microinjections mixture for zebrafish embryos


    Microinjection and expression of UAS construct (Day 0)

    1. The following morning (Day 0), remove the divider and allow approximately 15-20 min of undisturbed mating time (Video 1).

    2. Use a Pasteur pipette to align the embryos along the trenches and remove excess water to prevent embryos from moving during injection.

    3. Inject one-cell stage embryos with ~5 pL of DNA + RNA mixture under a stereoscope equipped with the injection apparatus. The apparatus consists of a needle holder attached to an MHC model magnetic stand and connected to PLI-90 Pico-injector, a foot switch to pulse the injection solution into the embryos and assisted by a backpressure unit to gauge the pulse release of pressurized nitrogen. Aim to inject around 200 to 300 embryos in 1 hour.

    4. Immerse injected embryos in E3 (1x). Save and raise 20 to 30 non- injected embryos alongside the injected embryos to assess microinjection induced damage. Use standard zebrafish embryos bleaching protocol (https://zfin.org/zf_info/zfbook/chapt1/1.5.html) and split them into 2 groups after bleaching.

    5. Incubate all the embryos at 28 °C in the dark. Use 1 group of the injected embryos for light exposure and the other as controls (dark).


  4. Fluorescence microscopy to detect mCherry expression in D. rerio

    1. Sort injected embryos under fluorescence stereomicroscope at 24 h post fertilization (hpf) (Day 1) for mCherry expression. Clear dead embryos from injected and uninjected dishes and change the water. Expect 5-10% injected embyos to die by 24 hpf.

    2. Replenish the embryos with fresh E3 (1x) daily until they reached 5 days post fertilization (dpf). Send larval zebrafish with mCherry expression to be grown in separate tanks in the hatchery from the non-expression larvae by 7 dpf.

    3. Cover the lid of the container used to grow baby fish with aluminum foil to prevent direct light exposure. Covering can be removed when fish reach adulthood depending on the expression tissue (for instance, sparse neuronal expression)

    4. To generate a permanent line, genotype adult fish by fin clipping at 8 weeks post fertilization (wpf). Use primers specific for Aβ-CRY2 and Gal4 for genotyping. Cross fish which are positive for both Aβ-CRY2 and Gal4 to obtain F1 embryos. Screen embryos for mCherry expression from 4 dpf onwards and follow the steps above. Tol2 transgenesis usually yields more than 50% transgene. However, due to the properties of the Abeta-CRY2, we found only 10% of the injected fish carries both Aβ-CRY2 and Gal4 at the point of genotyping. And only 1-2% showed germline transmission.

      Note: It is possible to find fish which are positive for both Gal4 and Abeta-CRY2 in genotyping but do not express mCherry. This could be due to the silencing of the UAS- regulated effector/reporter genes by methylation at the CpG nucleotides (Akitake et al., 2011) or may also due to other unknown factors such as the genomic location of the insertion.


  5. Light-sheet microscopy to image neural development during embryogenesis (in D. melanogaster)



    Figure 2. Bleaching and mounting of Drosophila embryo into glass capillary for long term imaging. A. Place a stage 10 embryo in the middle of a piece of C-fold towel and add bleach till soaked. Rotate the embryo to encourage dechorionation. After bleaching, move the C-fold with the embryo on it to a drying C-fold towel using forceps (i). Rinse the embryo with water by transferring the piece of C-fold to a drop of water, let it dry once again on the drying C-fold (ii), and rinse once more in another drop of water before leaving it to dry on the drying C-fold towel (iii). B. Place a capillary filled with melted agarose and hold it in place gently on top of an inverted petri dish. Pick the bleached embryo from the C-fold and place it on the surface of the agarose. C. Push the embryo gently into the agarose using the fine tip of a copper wire and position it parallel to the capillary. D. After allowing the agarose to solidify for 5 min, load the capillary into the sample holder, and lower it into the sample chamber of the Light-sheet Z.1. Eject the agarose slowly such that the embryo just emerges from the capillary. Position the embryo in front of the objective such that the ventral surface (v) faces the objective. d: dorsal. These steps are shown in Video 2.


    Video 2. Mounting and imaging of Drosophila embryos


    1. Melt a tube of 1% low gelling temperature agarose at 70 °C for half an hour in a heat block and then cool it to 37 °C, mix thoroughly by inverting before proceeding with mounting.

    2. Remove the grape agar juice plate from a cup that has been laying eggs for at least 10 h to ensure you obtain a good amount of embryos at stage 10 (Hartenstein, 1993). Pour approximately 1 ml of Halocarbon oil 27 onto the plate with embryos and swirl the oil in a circular manner to spread it out evenly and form a thin layer of oil on the plate. Let it rest for 1 min to allow the oil to penetrate the chorion of all the embryos.

      Note: It is best to use plates that have flies laying eggs for 24 h so that proper development and expression of the GFP and mCherry fluorescent proteins in older embryos can be observed before proceeding with mounting and imaging of stage 10 embryos which do not express high levels of these fluorescent tags.

    3. Observe the plate under a fluorescence microscope using transmitted light.

    4. After germ band retraction (stages 14-17 according to [Campos-Ortega and Hartenstein, 1997]), embryos should show a very bright GFP and mCherry signal in the nervous system that mark the glial cells and neurons respectively using reflected light.

    5. Pick 1 stage 10 embryo from the plate using a dissecting needle and transfer it to the center of 1 cm x 1 cm piece of C-fold in a Petri dish under the transmitted light (Figure 2A).

    6. Add a drop of bleach to just soak the piece of C-fold. Add 2 drops of autoclaved water in the same Petri dish as shown in Figure 2A. Use the dissecting needle to roll the embryo up and down on the C-fold and encourage the chorion to peel off. Within 30 s to a minute, the chorion can be seen peeling off under the transmitted light at high magnification with the dorsal appendages disappearing and the matt embryo surface will take on a glossy finish.

    7. As soon the chorion is removed, use dissecting forceps to carefully lift the C-fold from one corner with the dechorionated embryo on it to a dry big piece of C-fold to remove as much bleach as possible (Figure 2A(i)). Rinse the embryo with water by moving the C-fold to the first drop of water, allowing it to soak for 30 s, then move it to another region on the drying C-fold (Figure 2A(ii)). Repeat the rinse with water one last time and allow the C-fold to dry as much as possible by allowing it to rest on an un-used part of the drying C-fold for a minute (Figure 2A(iii)).

    8. Meanwhile, insert the plunger into the size 2 glass capillary and suck up the melted 1% low-melting agarose Type VII-A (Sigma) at 37 °C to just above the black line as shown in Figure 2B. Position the capillary on top of an inverted Petri dish and allow the agarose to cool in the capillary for a minute.

    9. Pick up the dried embryo from the C-fold using a dissecting needle and position it in the center of the capillary (Figure 2B).

    10. Push the embryo into the center of the solidifying agarose using a fine copper wire to a depth of approximately 0.5 cm and maneuver it such that it is parallel to the capillary (Figure 2C). Remove the copper wire very slowly from the agarose once the embryo is in the desired position. Wait for 5 min to allow the agarose to solidify.

      Note: Align the embryo in the center of the capillary to ensure it is held securely in the agarose. If it sticks to the glass capillary, push it back into the agarose to avoid slipping out during imaging. This step requires a lot of patience and practice. This was the best way to position the embryo to obtain the highest resolution images. The aliquot of 1% low-melt agarose can be left at 37 °C for use over a long time or cooled and melted when needed several times. However, it will eventually stop solidifying after an extended period of time at 37 °C or after numerous heat-cool cycles. This requires the aliquot in use to be discarded and a new aliquot used thereafter.

    11. Switch on the Light-sheet Z.1 system and start up the Zen 2014 SP software. Inject autoclaved water into the sample chamber such that it filled to the brim as shown in the manufacturer’s protocol.

    12. After the agarose in the capillary has solidified, load the capillary into the sample holder according to the manufacturer’s manual and secure it by tightening the screw. Load the sample holder into the Light-sheet Z.1 and lower it into the sample chamber filled with autoclaved water. Push the embryo out of the capillary slowly such that it is just outside capillary and aligned with the objective (Figure 2D).

    13. Use the Light-sheet Z.1 10x/0.2 Illumination Optics to rotate the capillary such that the ventral surface (v) of the sample faces the objective to obtain high resolution images of the ventral surface (Figure 2D). Acquire images using a Light-sheet Z.1 Illumination objective 40x/1.0 (water immersion). Set up the 20 mW 561 nm laser at 13% laser power with 12.5 ms exposure time and the 30 mW 488 nm at 10% power with 29.95 ms exposure time to activate CRY2 clustering. Set z-stacks at 1 μm intervals and denote the top of the z-stack as approximately 20 sections (20 μm) above the top of the embryo and the bottom of the z-stack as approximately 20 sections (20 μm) from the bottom of the embryo. This is to ensure data is not lost in case the embryo shifts during imaging. Excess z-stack sections can be removed during processing. Set-up a time series acquisition with dual-side illumination in both channels every 2.5 min for 500 cycles. For embryos not exposed to blue light, use only the 561 nm laser. Allow the image acquisition to run for approximately 22 h.

    14. After the imaging is completed, pull the plunger up to insert the embryo back into the capillary and remove the sample holder from the Light-sheet Z.1. Eject the embryo with the agarose and discard these. The capillary can be reused several times.


  6. Light-sheet microscopy to image neuronal damage (in D. rerio)

    1. A custom designed open light-sheet microscope was used for acquiring videos of the zebrafish larvae.

    2. Briefly, mount samples in a customized chamber for light-sheet microscopy on a horizontal stage controlled by an ultrasound motorized 10 nm step size Z-Drive.

    3. Generate a light-sheet using a single illumination arm in front of the sample mounting stage delivering laser beam (488 nm or 561 nm) through a 10x illumination objective.

    4. Acquire images on a Hamamatsu Orca Flash 4.0 V3 sCMOS Camera through a Super High NA (1.0; XLUMPLFLN20xW) 20x water dipping lens.

    5. Use multidimensional acquisition in MetaMorph software with streaming option to acquire data.

    6. Use an acquisition protocol to acquire 1 z-stack with 2  μm step-size per min for 30 min.

    7. Illuminate the sample by 561 nm laser light filtered through a Texas Red/mCherry/AlexaFluor 594 dichroic. At minutes 5 and 6, flood the stage with high intensity transmission white light from X-Cite 120LED System for 45 s. Resume the time lapse imaging for the next 25 min.

    8. Repeat the whole process a second time after acquisition of the first time-lapse.

    9. Make videos from time-lapse images.


  7. Danio rerio Mitochondrial metabolic flux assay

    1. Split injected embryos (C) into 2 dishes for 48 h; one for light exposure condition, and the other as the dark control.

    2. Dechorionate both dishes with minimal reflected light.

    3. Expose experimental embryos to high intensity LED light on a stereoscope for 2 h. Embryos are then loaded into the 96 well plate using a fine Pasteur pipette.

    4. Measure Oxygen Consumption Rate (OCR) using the XF96 Extracellular Flux Analyzer.

    5. Place one embryo (48 hpf) in 175 μl of E3 water in each well and start the experiment.

    6. Measure OCR every 8 min for 6 cycles (48 min), then add a drug.

    7. Calculate the volume of injection needed from the stock concentration of each drug, such that the final concentrations in the well are – a) 9.4 M oligomycin, b) 2.5 μM FCCP and c) 20 mM sodium azide.

    8. The experiment is summarized in Figure 3.



      Figure 3. Schematic of metabolic flux assay on transiently expressing Aβ-CRY2-mCherry embryos


  8. D. rerio Adenosine triphosphate (ATP) assay

    1. Use the remaining embryos from (Procedure C) for ATP assay. Or repeat Procedure C to generate more embryos.

    2. Pool five 48 h embryos in 1.5 ml Micro tubes and add 50 μl ice-cold 10% TCA buffer. For each condition make at least 8 such pools (n = 8).

    3. Use a hand held micropestle homogenizer, to homogenize embryos on ice.

    4. Centrifuge the homogenate at 4 °C at 10,000 RPM for 5 minutes.

    5. Transfer the supernatant to a new tube and keep at -80 °C for use later, or use immediately.

    6. Add 5 μl of sample supernatant or ATP standards into a white 96-well microtiter plate (Figure 4).

    7. Measure ATP luminescence using BioTek cytation 3 plate reader preprogrammed to inject arsenite ATP buffer (150 μl/well) followed by firefly lantern extract (45 μl/well).



      Figure 4. Schematic shows the summary of procedures using zebrafish


  9. Fluorescence microscopy to visualize Aβ aggregates in C. elegans

    1. Wash nematodes off the plates into an Eppendorf tube.

    2. Add 1 mM Levamisole into the worm suspension.

    3. Gently mix the worm suspension and place 20 μl of the worm suspension onto a glass slide containing 2% agarose [see Verbrugghe and Chan (2011) on how to make agarose pad].

    4. Switch off ambient light and bring the slides for imaging using 561 nm and 488 nm laser.

    5. Using 488 nm laser, select pharyngeal-GFP positive nematodes for the transgenic animals.

    6. Using 561 nm laser, observe the A&Bgr; expression and aggregate levels.


  10. Other assays used to confirm aggregation-induced deficits in C. elegans

    1. Lifespan assay (He, 2011), mitochondrial metabolic flux assay (Ng and Gruber, 2019) and ATP assay (Schaffer et al., 2011) can be used to confirm A&Bgr; aggregation-induced deficits.

Data analysis

  1. Light-sheet microscopy to image neural development during embryogenesis

    1. Process the acquired images file in the ZEN 2014 SP software.

    2. The embryo is likely to shift slightly during imaging. Hence, determine the z-stack section that includes the top of the embryo at every time point (ZT) (Figure 5). Thereafter, note the z-stack section (ZB) in the embryo that stays in focus throughout the time points after which the focus is lost (Figure 5 shows an example of the top, bottom and middle z-stack images for the control embryos which was used to subsequently generate Video 3 in the original research paper [Lim et al., 2020]). Create a subset file including the images from ZT to ZB.

    3. If the embryo does not show any changes in development after a certain time point (T1), create a further subset from the start of imaging to T1.

    4. Create a maximum intensity projection of the final subset file and adjust the display settings that best suit the image. Export the time series as a movie in the format of choice.



      Figure 5. Annotation of the top (ZT) and bottom (ZB) z-stack of the embryo during post-processing of the data file to create a subset file. ZT is defined as the top of the embryo at every time point (ZT) and ZB is the z-stack at which the embryo is in focus throughout the time points after which the focus is lost. Scale bar shows 50 μm.


  2. D. rerio Mitochondrial metabolic flux assay (Figure 4)

    1. Use the Seahorse Wave Controller software, compute the following area under the curve (AUC):
      AUC1 = oxygen consumption rate measurement 1 to 6 (pre-injection)
      AUC2 = oxygen consumption rate measurement 7 to 12 (after oligomycin injection)

      AUC3 = oxygen consumption rate measurement 13 to 18 (after FCCP injection)
      AUC4 = oxygen consumption rate measurement 19 to 24 (after sodium azide injection)

    2. Determine the respective respiration rate as follow:
      Basal respiration = AUC1 – AUC4

      Proton leak = AUC2 – AUC4
      ATP-linked respiration = AUC1 – AUC2
      Maximum respiration = AUC3 – AUC4
      Spare respiratory capacity = AUC3 – AUC1
      Non-mitochondria respiration = AUC1 – AUC4

    3. Plot the individual respiration rate in bar graph and analyze the group mean using one-way ANOVA.


  3. D. rerio ATP assay

    1. Calculate ATP level for all samples using the standard curve constructed from the ATP standard.

    2. Compute the mean ATP level for different conditions and analyze the data using one-way ANOVA.

Notes

For sample imaging in the Light-sheet Z.1 for D. melanogaster, the sample chamber heats up significantly due to the short time interval of 2.5 min. Hence, the room temperature was set to 18 °C to prevent over-heating of the water in the chamber that prevents development of the embryo. Alternatively, the water can be cooled by setting up a peristaltic pump attached to the sample chamber.

Recipes

  1. Standard Fly food

    6 g Bacto agar

    114 g glucose

    56 g cornmeal

    25 g Brewer’s yeast

    20 ml of 10% Nipagin in 1 L final volume

  2. Grape juice agar plates

    Prepare grape juice agar using FlyStuff Grape Agar Premix, For Embryo Collection (25 Packets/Unit) according to the manufacturer’s instructions and pour approximately 4-5 ml of agar per 60 mm Petri dish. Allow to solidify and store the plates at 4 °C.

  3. Yeast paste

    Mix yeast (Bruggeman Instant Dry Yeast Blue, 500 g) with a small amount of water in a plastic beaker to achieve a thick paste. The yeast paste can be kept in a beaker covered with aluminum foil at 4 °C.

  4. 1% low gelling temperature agarose in water aliquoted into 1.5 ml Eppendorf tubes

    1. In a 50 ml microwave-safe glass, add 0.1 g of Agarose, low gelling temperature Type VII-A and add 10 ml of autoclaved distilled water

    2. Microwave with intermittent mixing till the agarose just dissolves without boiling to prevent excessive loss of water

    3. Once the agarose has dissolved completely, let the mixture cool slightly and aliquot approximately 1 ml of the 1% agarose into 1.5 ml Eppendorf tubes

    4. Close the Eppendorf tubes after the agarose has solidified and store at room temperature

    5. For mounting, melt a tube of 1% low gelling temperature agarose at 70 °C for half an hour in a heat block and then cool it to 37 °C, mix thoroughly by inverting before using for mounting

  5. 2% low gelling temperature agarose in water aliquoted into 1.5 ml Eppendorf tubes (for C. elegans imaging)

    Same as above, but dissolve 0.2 g of agarose, low gelling temperature Type VII-A in 10 ml of autoclaved distilled water

  6. 60x E3 (embryo medium) stock solution (for D. rerio)

    1. Dissolve 172 g NaCl, 7.6 g KCl, 29 g CaCl2·2H2O and 49 g MgSO4·7H2O in 10 L of Milli-Q water

    2. Dilute 160 ml of 60x stock solution in distilled water to make up to 10 L of E3 (1x)

  7. 2 % low melting agarose

    1. Dissolving 1 g of agarose powder in 100 ml E3 (1x)

    2. Cook in microwave for 2 min at low power (for fish imaging)

  8. 25x Tricaine stock solution (for D. rerio)

    1. Dissolving 400 mg Tricaine in 97.9 ml DD water

    2. Adjust pH to 7 using Tris-HCl (1 M)

    3. To use Tricaine for anesthesia, dilute stock solution by adding 96 ml E3 (1x) to 4 ml of 25x stock solution in a beaker

    4. Diluted Tricaine should be disposed in activated charcoal

  9. 10% TCA buffer

    Dissolve 1 g TCA powder in 10 ml distilled water

  10. Arsenite ATP buffer

    Mix 80 mM MgSO4·7H2O

    10 mM KH2PO4

    100 mM Na2HASO4·7H2O in 1:1:1 ratio

Acknowledgments

We are thankful for the funding provided by Ministry of Education Singapore AcRF grant IG17-LR005, IG17-BS101 and IG18-BS002 to JG, Yale-NUS College grant R-607-265-225-121 to ASM, AcRF grants IG17-LR006 and IG18-LR001 to NST.

Competing interests

We have no conflicts of interest to declare.

Ethics

Institutional biosafety and genetic manipulation guidelines of IMCB were followed for generation of transgenic zebrafish. Fish husbandry, rearing and maintenance were performed following approved protocols by Institutional Animal Care and Use Committee (IACUC) of the Biological Resource Center at A*STAR. Approved experimental protocols (IACUC 191501) were followed.

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简介

[摘要]Alzheimer'sdisease(AD)长期以来与淀粉样前体蛋白产生的细胞外淀粉样斑块(Aβ)的积聚有关。然而,在健康人身上发现了斑块,并不是所有的AD大脑都有斑块,这表明细胞外Aβ聚集体的作用可能比预期的要小。在疾病进展过程中研究Aβ肽的一个局限性是无法以可控的方式诱导聚集。我们开发了一种诱导Aβ聚集的光遗传学方法,并在三种模式生物中测试了其生物学效应:D.melanogaster、C.elegans和D.rerio。我们产生了一个荧光标记的,光生的

一种β肽,在所有生物体内,在蓝光存在下迅速寡聚。在这里,我们详细介绍了在动物模型中表达该融合蛋白的程序,使用延时光片显微镜研究对神经系统的影响,并进行代谢分析来测量由于细胞内Aβ聚集而引起的变化。这种方法利用光遗传学来研究AD的病理学,实现了目前任何其他方法都无法实现的体内时空控制。

[背景]阿尔茨海默病(AD)是一种衰弱的、与年龄相关的神经退行性疾病(Zhang等人,2011年;De Paula等人,2012年;Kumar等人,2015年)。淀粉样β(Aβ)作为细胞外Aβ斑块的积累被认为是该病的一个主要原因(Hardy和Higgins,1992),但是针对这些Aβ斑块的干预措施的失败及其在无AD症状个体大脑中的存在(Hawkes,2016;Cummings等人。,2018)建议需要进一步的机械分析。有人提出了由可溶性细胞内Aβ聚集物诱导的神经毒性的另一种假说(LaFerla et al.,2007;Ferreira and Klein,2011),但目前缺乏在体内控制Aβ寡聚化的工具来研究可溶性Aβ聚集体对疾病进展的影响和药物的治疗潜力。

光遗传学是实现这一目标的理想工具,因为它允许生物体在特定波长的光下,对体内蛋白质进行高度精确的时空调控

(Moglich和Moffat,2010年;Fenno等人,2011年)。我们已经将这种方法应用于包括胚胎发育和体内平衡在内的各种问题(Kaur等人,2017年;Bunnag等人,2020年),并且最近发现,利用光遗传学诱导可溶性aβ蛋白聚集导致代谢和物理损伤,并恶化3种模式生物——果蝇的寿命和健康寿命melanogaster,C.elegans和D.rerio,以及标准组织培养模型(Lim和Mathuru,2017;Lim等人,2020;)。在这里,我们描述了一种用于构建光致发光诱导物的方法

一种β蛋白,在不同的模型生物体内暴露于蓝光时寡聚,并通过各种代谢测定和延时实时成像研究其下游的有害影响。我们使用拟南芥隐色素2的光裂合酶同源区作为一种光发生开关(CRY2)蛋白,在蓝光存在下快速可逆地寡聚成光体(Mas等人,2000年;Bugaj等人,2013年)。这个CRY2结构域被生成为aβ-

CRY2-mCherry融合蛋白在蓝光激活下诱导Aβ蛋白聚集,mCherry荧光蛋白可见。

将这种多功能的工具与D.melanogaster等易于操作的遗传模型相结合,

C、 挽歌和D.rerio,给了我们暂时控制可溶性Aβ寡聚体的诱导,这在其他现有的阿尔茨海默病动物模型中是不可行的。我们还能够使用实时成像和成年苍蝇实时解剖Aβ寡聚体对胚胎发育过程中的病理影响(Lim等人,2020年)。这个模型还允许我们生成胚胎的马赛克,使得只有一半的胚胎,或一小部分暴露在蓝光下。因此,我们可以研究靶向光诱导的Aβ寡聚化对各种解剖结构的影响,例如以胚胎的未发光部分为对照的神经系统。在无脊椎动物和脊椎动物实验室模型中的功能性证明了该技术在研究诱导Aβ寡聚化的生物化学、代谢、细胞和神经生物学方面的应用。潜在的药物干预对寿命、健康寿命和新陈代谢的影响也可以用这种强有力的光遗传学方法在各种模式生物中进行研究。我们能够采用不同的策略在无脊椎动物和脊椎动物中表达相同的转基因,这应该通过条件敲入法转化为小鼠研究。用蓝光诱导成年小鼠脑内聚集,研究Aβ-CRY2-mCh聚集对神经元功能和行为的影响。这种方法对Aβ有特异性,但只要改变起始基因,就可以很容易地适应其它聚集蛋白。

关键字:光遗传学, 淀粉样蛋白-β, 阿尔兹海默症, 光片, 黑腹果蝇, 斑马鱼, 秀丽隐杆线虫


材料和试剂
A、 光生转基因的分子克隆
1. 10 cm培养皿(Greiner Bio One International,目录号:633185)
2. 14毫升聚丙烯管(生物实验室,目录号:352059)
3. 1.5 ml微管(Axygen,目录号:MCT-150-L-C)
4. 移液管尖端(适用于100-1000μl、20-200μl、2-50μl和0.5-10μl移液管)
5. 微型装载机(Eppendorf)
6. pUCIDT-attL1-人类ABeta-attR5(地址:160436)
注:Aβ人类基因由attL1和attR5悬置合成卡那霉素耐药质粒主干,用作网关输入载体。 
7. pUCIDT-attL1-蠕虫ABeta-attR5(Addgene:160437)
注:Aβ蠕虫基因由attL1和ATRT5悬置合成卡那霉素抗性质粒主干,用作网关输入载体。
8. pDONR-attL5-CRY2-mCh-attL2(地址:160438)
注:CRY2 mCh是从pCRY2PHR-mCherryN1(Chandra Tucker的礼物(Addgene质粒,目录号:26866)(Kennedy等人,2010年)。attB5和attB2位点被添加到CRY2 mCh PCR产物中,并重组到pDONR P5-P2载体中。最终的pDONR-attL5-CRY2-mCh-attL2质粒作为网关进入载体。
9. pUASg HA attB(果蝇基因组学资源中心,库存编号:1423)
注:采用的载体特异性测序引物为HSP
转发:Tataatagagggcgcttcgt
HA反面:AGGTAATCTGGAACGTATA
10.  pDEST-hsp16-2p(东京医学研究所黑名秀彦赠送的礼品)
医学与牙科大学)
注:采用的载体特异性测序引物为Hsp16-2p:CGAATGTGATCGCCCTCCT
11.  pDEST-Tol2-PA2-CMV(Tol2套件)。用于以下Danio rerio转基因的分子克隆:a)pDEST-Tol2-PA2-10xUAS-aβ-CRY2-mCh;b)pDEST-Tol2-PA2-Ubi-aβ-CRY2-mCh;c)pDEST-Tol2-PA2-CMV-aβ-mCh(Addgene ID:160435)
12.  前10名合格电池(赛默飞世尔科技公司,目录号:C404003)
13.  琼脂糖,生物技术级,500 g(第一个碱基,目录号:BIO-1000-500g)
14.  LB琼脂磨碎机,细菌培养基,500 g(1基,目录号:BIO-4010-500g)
15.  LB肉汤米勒,pH 7.0,细菌培养基,500 g(第一个碱基,目录号:BIO-4000500g)
16.  氨苄西林钠盐(西格玛,目录号:A9518G)
17.  硫酸卡那霉素(西格玛,目录号:K1377)
18.  MultiSite Gateway®Pro Plus套件(目录号:12537100)
19.  GatewayTM LR ClonaseTM II酶混合物(Thermo Fisher Scientific,目录号:11791100)
20.  GatewayTM BP克隆酶混合物(Thermo Fisher Scientific,目录号:11789020)
21.  Phusion高保真PCR试剂盒(Thermo Fisher Scientific,目录号:F553S)
22.  Gibson Assembly®克隆套件(新英格兰生物实验室,目录号:E5510S)23。低熔点琼脂糖(Promega,目录号:V2111)
 
B、 光生转基因在体内神经元中的表达(黑腹锦鸡)
1. 1L微波安全玻璃烧杯
2. 磁力搅拌器
3. 50毫升玻璃烧杯
4. 铲
5. 压缩二氧化碳气瓶(任何品牌)
6. LED灯(任何品牌)
7. 铝箔(任何品牌)
8. 泡沫塑料盒(任何品牌)
9. 用于60 mm培养皿的蝇笼(任何,例如用于60 mm培养皿的小型胚胎收集笼,苍蝇,目录号:59-100)
10.  60mm培养皿(任何品牌,如NuncTM EasYDishTM培养皿,ThermoFisher Scientific,目录号:150462)
11.  果蝇分类刷(任何品牌)或非常薄的油漆刷
12.  果蝇[布卢明顿果蝇库存中心(NIH P40OD018537)]。库存编号458,elav促销员为GAL4/UAS系统驱动GAL4(Brand和Perrimon,1993)
13.  P(ET-QF2.GU)回购[Q系统来自布卢明顿果蝇股票中心(NIH P40OD018537),股票编号:66477](Lin and Potter,2016)
14.  P(QUS-mCD8-GFP)X(布鲁明顿果蝇库存中心的Q系统
P40OD018537),库存编号:30001)(林和波特,2016年)
15.  细菌用琼脂
16.  葡萄糖
17.  玉米粉
18.  酿酒酵母19。尼帕金
20.  FlyStuff葡萄琼脂预混料,胚胎采集用(25包/单位)(FlyStuff,目录号:GEN47-102)
21.  酵母糊(布鲁格曼速溶干酵母蓝,500g)
22.  标准苍蝇食品(见食谱)
23.  葡萄汁琼脂板(见配方)
24.  酵母糊(见配方)
 
C、 结构的交叉和表达(对于D.rerio)
1. 3毫升巴斯德吸液管(Tarson,目录号:T940050)
2. Tg gng8:Gal4型(感谢Marnie Halpern教授)(Hong等人,2013年)
3. 甘油三酯无人机:GCamp6S在珍珠层背景中(mitfa-/-)
4. 三卡因(MS-222或3-氨基苯甲酸乙酯甲烷磺酸盐;Sigma,目录号:A5040)
5. 氯化钠
6. 氯化钾
7. CaCl2·2H2O
8. 硫酸镁·7H2O
9. 盐酸三氯氢钠
10.  25x三胺原液(用于D.rerio)
11.  配方(见第60x号储备溶液)
D、 UAS构体的微注射与表达(用于D.rerio)
1. 带灯丝的标准壁硼硅酸盐玻璃(萨特仪器,型号:外径1mm,内径0.5mm,长度7.5cm,目录号:BF100-5-7.5)
 
E、 光片显微术在胚胎发生过程中的神经发育成像(用于黑腹锦鸡)
1. 一次性滴管(任何品牌)
2. 15毫升猎鹰管(任何品牌)
3. 不锈钢解剖针,L160 mm(Hammacher,HWO010-16,目录号:912483)
4. 玻璃毛细管,尺寸2黑色,毛细管内径~1 mm(品牌GmbH,目录号:701932),带有相应的柱塞
5. 5厘米长的铜线(或其他细线)
6. 称重船
7. 卤代烃油27(化学原料有限公司,目录号:3904 9000)
8. 漂白剂(克罗克斯)
9. 高压灭菌密力克水
10.  1%琼脂糖,低胶凝温度VII-A型(Sigma,目录号:A0701),溶于水中,等份加入1.5 ml Eppendorf试管中(见配方)
 
F、 鱼类神经元Aβ-CRY2-mCh的光片显微术研究(英文)
1. 安装介质加热块
 
G、 线粒体代谢通量测定斑马鱼
1. 寡霉素(Sigma-Aldrich,目录号:75351)
2. 羰基氰化物4-(三氟甲氧基)苯腙(FCCP)(Sigma-Aldrich,目录号:C2920)
3. 叠氮化钠(Sigma-Aldrich,目录号:C2002)
4. 二甲基亚砜(目录号:二甲基亚砜650)
 
H、 三磷酸腺苷(ATP)测定斑马鱼
1. 萤火灯提取物(Sigma-Aldrich,目录号:FLE250)
2. MgSO4·7H2O(Sigma-Aldrich,目录号:230391)
3. KH2PO4(Sigma-Aldrich,目录号:P0662)
4. Na2HASO4·7H2O(Sigma-Aldrich,目录号:S9663)
5. TCA粉末(Sigma-Aldrich,目录号:T6399)
6. 10%三氯乙酸(TCA)溶液(见配方)
7. 亚砷酸盐ATP缓冲液(见配方)
8. 25x三胺原液(用于D.rerio)(见配方)
 
一、 荧光显微镜观察线虫Aβ聚集体
1. 1.5 ml Eppendorf管(任何品牌)
2. 盐酸左旋咪唑(西格玛,目录号:196142)
3. 溶于水中的2%琼脂糖(Sigma,产品编号:A0701)等分入1.5 ml Eppendorf试管中(见配方)
4. 2%低熔点琼脂糖(见配方)
 
设备
 
A、 光生转基因在体内神经元中的表达(黑腹锦鸡)
1. 果蝇带CO2垫的工作站(Flowbuddy完成;Genesee Scientific,目录号:59-122WC)
2. 25°C苍蝇孵化器(任何品牌,如INVICTUS Drosophila孵化器、Genesee Scientific)
3. 立体显微镜(任何品牌,如徕卡,型号:M50)
4. 荧光立体显微镜(任何品牌,如徕卡MZ16F)
 
B、 光片显微术在胚胎发生过程中的神经发育成像(用于黑腹锦鸡)
1. 细尖锯齿解剖钳(任何品牌)
2. 温度范围为37°C和70°C的加热块(任何品牌,如数字块加热器、赛默飞世尔科学公司)
3. 蔡司灯片Z.1(卡尔蔡司)
4. 样品室
 
C、 用于D.rerio的实验
1. 细菌培养箱,37°C
2. 细菌培养摇瓶培养箱,37°C
3. 斑马鱼Aβ聚集的薄层显微术研究
4. 海马细胞外通量分析仪(安捷伦科技,海马生物科学,型号:XFe96)
5. Cytation 3细胞成像多模式阅读器(BioTek仪器)
6. 微杵均质机
 
D、 荧光显微镜观察线虫Aβ聚集体
1. 斑马鱼胚胎培养箱,28°C
2. 镊子(F.S.T,型号:Student精细镊子,直形,目录号:91115-10)
3. 微量移液器(萨特仪器,型号:P-1000)
4. 微注射系统(哈佛仪器,型号:PLI-90 Pico注射器)
5. 共聚焦显微镜(蔡司,型号:LSM800)
 
E、 用于确认线虫聚集诱导缺陷的其他试验
1. 培养箱(水溶性,TC 255 S)
2. 海马细胞外通量分析仪(安捷伦科技,海马生物科学,型号:XFe96)3。Cytation 3细胞成像多模式阅读器(BioTek仪器)
 
软件
 
光片显微术在胚胎发生过程中对神经发育的成像
1. ZEN 2014 Light sheet Z.1处理软件包(卡尔蔡司,目录号:410136-1059-130)
2. 变形®显微镜自动化和图像分析软件(分子器件)
 
程序
 
A、 光生转基因在黑腹锦鸡和秀丽隐杆线虫中表达的分子克隆
利用人Aβ1-42氨基酸序列(Aβ人)为果蝇和斑马鱼产生转基因。对于线虫,使用aβ1-42(aβ蠕虫)的线虫密码子优化版本(Fong等人,2016年)。对于人类Aβ-CRY2 mCh的果蝇表达,在网关输入载体(pUCIDT-attL1-human abetattr5)和pDONR-attL5-CRY2-mCh-attL2中获得带有attL1和attR5悬置的人类AβDNA序列。根据制造商的说明(Invitrogen,ThermoFisher Scientific),执行多站点网关LR反应,将这些结构插入pUASg HA attB果蝇表达载体(Bischof等人,2007)。对于秀丽隐杆线虫Aβ-CRY2 mCh的表达,在pUCIDTattL1蠕虫ABeta-attR5输入向量、pDONR-attL5-CRY2-mCh-attL2和pDEST-hsp-16之间执行多站点网关LR反应
2p C.elegans表达载体,按照制造商的说明(Invitrogen,ThermoFisher Scientific)。对于所有产生的转基因,使用载体特异性引物进行测序,以确定两个片段的方向和插入目的载体。
 
B、 斑马鱼(斑马鱼)光遗传学转基因的分子克隆——a)pDEST-Tol2-PA210xUAS-aβ-CRY2-mCh;B)pDEST-Tol2-PA2-Ubi-aβ-CRY2-mCh;c)pDEST-Tol2-PA2-CMV-aβmCh
将感兴趣的单个DNA元件克隆到网关兼容载体中
1. PCR扩增10xUAS、Ubi和CMV序列,用特异性引物attB4(正向引物)和attB1(反向引物)克隆到pDONRTMP4-P1R中,经BP重组生成5'进入质粒。通过测序引物M13F或5‘GTAAACGAGGCCAGT3’和M13Rev 5‘CAGGAAGCTATGAC3’来验证克隆是否正确。
2. 用特异性引物attB1(正向引物)和attB2(反向引物)侧翼特异性引物扩增Aβ-CRY2 mCh,并克隆到pDONR221TM中,经BP重组产生中间入口质粒。用M13引物(与1相同)验证克隆是否正确。
注:含有poly-A信号的3'进入载体(p3'PolyA)和含有Tol2识别位点的目的质粒(pDestTol2PA2)起源于川崎实验室,可作为–Tol2工具包(Kawakami,2007年)。
3. 在BP重组后,在每个进入质粒上创建兼容的attL位点。LR重组生成表达质粒。通过测序引物验证克隆的正确性
前进档:5'CAAGTTGTACAAAAAGCGCTTA3'倒档:5'TACCAGCTTCTTGTACAAAGGGGGGA 3'。
4. 图1显示了片段组装的摘要。
 
 
图1。斑马鱼转基因表达克隆的构建
 
C、 光生转基因在神经元中的表达
对于D.melanogaster,将pUASg humanAβ-CRY2 mCh HA attB转基因注入attP2(菌株#8622)P[CaryP]attP268A4(BestGene Inc(加利福尼亚))(Groth等人,2004;Markstein等人,2008)。如有要求,将提供转基因苍蝇。分离至少5只纯合子雄性UAS-AβCRY2 mCh果蝇,并与至少10只Elav-Gal4,Repo-QF2;QUAS-GFP雌性处女苍蝇杂交,以驱动Aβ-CRY2 mCh的神经元表达和GFP的胶质细胞表达。把这些雄性和雌性苍蝇放在蝇笼里,用涂有少量酵母糊的预热葡萄汁琼脂板覆盖,以鼓励产卵。将笼子放在25°C的培养箱中。控制苍蝇,使用UAS-td番茄纯合雄性代替。每天更换果蝇板,换上预热的葡萄汁琼脂板,上面有少量酵母糊。允许新搭建的笼子产卵约3天,以确保在使用它们进行实验之前有大量的发育中的胚胎。
对于秀丽隐杆线虫,从隐杆线虫遗传中心获得转基因菌株gnaEx1[myo-2p::YFP+hsp-16-2p::Abeta1-42::CRY2::mCherry])。通过次氯酸盐漂白使转基因线虫年龄同步(Stiernagle,2006)。为了诱导hsp16-2启动子驱动的βCRY2 mCh的体内表达,将第1天的成年线虫在35℃下孵育1h。培养1h后,将线虫分成光/暗组,并在20℃培养箱中生长。暗处理时,用铝箔包裹含有线虫的盘子,并将包裹好的盘子放入聚苯乙烯泡沫塑料盒中。将泡沫塑料盒放在培养箱架的顶部,以避免暴露在LED灯下。光处理时,将含有线虫的平板直接放在培养箱底部机架上的LED灯下。
对于D.rerio:斑马鱼系(Gal4;UAS)在特定的神经元或细胞中表达转基因。为了防止普遍表达的幼虫寡聚化,在黑暗的培养箱中培养注入的胚胎直到实验。尽量减少转移印版或分拣时的光照,以避免暴露在高LED灯下。对于线粒体流量分析或ATP测量的光处理,将幼虫放在立体镜的白色LED灯下2小时。表达受限的幼虫(如光片成像中使用的那些)对光照不太敏感,但仍在黑暗中饲养,直到成像,如下节所述。
 
UAS构建物的微注射和表达(第1天制备)
1注射前一天,用分隔器将鱼放置在繁殖箱中(视频1)。
 
 
视频1。斑马鱼微量注射
 
2. 用过滤器收集胚胎,用自来水冲洗干净,然后将它们转移到含有E3(1x)的10厘米培养皿中。用巴斯德吸管除去未受精的鸡蛋和碎片。
3. 用微量移液管拔出器制备注射针,并用微量加载器回填表(表1)中列出的3μl混合物。
4. 将针头固定在与微注射系统相连的微操作器上。
5. 用定制的带凹槽的塑料模具制作一个含有3%琼脂糖的微量注射板。
表1。斑马鱼胚胎微量注射混合物
核酸
构造
集中
质粒DNA
pHuc:Gal4
50纳克/微升
 
pDestTol2PA2 UAS:Aβ-CRY2 mCh
50纳克/微升
核糖核酸
Tol2转座酶
50纳克/微升
 
UAS结构的微注射和表达(第0天)
1. 第二天早上(第0天),取下分体器,并留出大约15-20分钟的不受干扰的交配时间(视频1)。
2. 用巴斯德吸管沿着沟槽对齐胚胎,并去除多余的水分,以防止胚胎在注射过程中移动。
3. 在配备有注射装置的立体镜下,用~5pl的DNA+RNA混合物注射一个细胞期胚胎。该装置包括一个针头夹持器,该针头固定在MHC型磁性支架上,并与PLI-90 Pico注射器相连;脚踏开关将注射溶液脉冲送入胚胎;在背压装置的辅助下,测量加压氮气的脉冲释放。目的是在1小时内注射200到300个胚胎。
4. 将注射的胚胎浸入E3(1x)。保存和培养20到30个未注射的胚胎和注射的胚胎一起评估微注射引起的损伤。使用标准斑马鱼胚胎漂白方案(https://zfin.org/zf_info/zfbook/chapt1/1.5.html)漂白后分成两组。
5. 所有胚胎在28℃黑暗中孵育。用一组注射的胚胎进行光照,另一组作为对照(黑暗)。
 
D、 荧光显微镜检测麦氏菌在雷氏菌中的表达
1. 在荧光体视显微镜下,于受精后24小时(第1天)对注射的胚胎进行分类,进行mCherry表达。从已注射和未注射的培养皿中清除死胚胎,并换水。预计5-10%的注射过的embyos会在24小时内死亡。
2. 每天用新鲜的E3(1x)补充胚胎,直到受精后5天(dpf)。将具有mCherry表达的斑马鱼幼体与未表达的斑马鱼幼体在孵卵室中用7dpf与未表达的幼体分开培养。
3. 用铝箔盖住用来养小鱼的容器盖,以防阳光直射。当鱼达到成年时,可以根据表达组织(例如,稀疏的神经元表达)去除覆盖物
4. 在受精后8周剪鳍,获得一个永久系
(wpf)。用Aβ-CRY2和Gal4特异性引物进行基因分型。用Aβ-CRY2和Gal4均阳性的杂交鱼获得F1胚胎。从4个dpf开始筛选mCherry表达的胚胎,并遵循上述步骤。Tol2基因的转化率通常在50%以上。然而,由于Abeta-CRY2的特性,我们发现只有10%的注射鱼在基因分型时同时携带Aβ-CRY2和Gal4。只有1-2%的人表现出种系传播。
注:在基因分型中可以找到对Gal4和Abeta-CRY2都呈阳性但不表达mCherry的fish。这可能是由于CpG核苷酸的甲基化使UAS调节的效应器/报告基因沉默(Akitake等人,2011年)也可能是由于其他未知因素,如插入的基因组位置。
 
E、 光片显微术在胚胎发生过程中的神经发育成像(在D.melanogaster)
 
 
图2。果蝇胚胎的漂白和植入玻璃毛细管进行长期成像。A、 把一个10级胚胎放在一块C形折叠毛巾中间,加入漂白剂,直到浸湿。旋转胚胎以促进胚胎的分泌。漂白后,用镊子(i)将带胚胎的C形折叠带移到干燥的C形折叠毛巾上。用水冲洗胚胎,将一片C形折叠片转移到一滴水中,让它在干燥的C形折叠片(ii)上再次干燥,然后在另一滴水中再次冲洗,然后在干燥的C形折叠毛巾(iii)上晾干。B、 将一根充满融化的琼脂糖的毛细管放在倒置的培养皿上,轻轻地将其固定到位。从C-折叠中取出漂白的胚胎,放在琼脂糖表面。C、 用铜丝的细尖将胚胎轻轻推入琼脂糖中,并使其与毛细管平行。D、 使琼脂糖凝固5分钟后,将毛细管装入样品架中,并将其降低到光片Z.1的样品室中。缓慢地排出琼脂糖,使胚胎从毛细血管中出来。将胚胎放在物镜前面,使腹侧表面(v)面向物镜。d: 背侧。这些步骤如视频2所示。
 
 
视频2。果蝇胚胎的安装与成像
 
1. 将1%低胶凝温度琼脂糖在70°C下在加热块中熔化半小时,然后将其冷却至37°C,在继续安装之前,通过倒置彻底混合。
2. 从哈滕斯坦的葡萄汁中至少取出10粒葡萄汁。将大约1ml的卤代烃油27倒入带胚的板上,并以圆形方式旋转油,使其均匀分布,在板上形成一层薄薄的油。静置1分钟,让油渗入所有胚胎的绒毛膜。
注:最好使用让苍蝇产卵24小时的平板,以便在对不表达高水平荧光标签的第10阶段胚胎进行安装和成像之前,可以观察到较老胚胎中GFP和mCherry荧光蛋白的正常发育和表达。
3. 用透射光在荧光显微镜下观察平板。
4. 胚带回缩后(根据[Campos-Ortega和Hartenstein,1997]的第14-17阶段),胚胎应该在神经系统中显示非常明亮的GFP和mCherry信号,分别用反射光标记胶质细胞和神经元。
5. 用解剖针从培养皿中取出1个10期胚胎,并在透射光下转移到培养皿中1 cm x 1 cm C形折叠片的中心(图2A)。
6. 加上一滴漂白剂,就可以把C型折叠布浸湿。在同一培养皿中加入2滴高压灭菌水,如图2A所示。用解剖针在C形折叠上下滚动胚胎,鼓励绒毛膜脱落。在30秒到1分钟内,在高倍透射光下可以看到绒毛膜脱落,背部附属物消失,无光泽的胚胎表面将呈现出光泽。
7. 一旦绒毛膜被切除,用解剖钳小心地从一个角落将C形折叠带从一角抬起,并将其放在一块干燥的C形折叠块上,以尽可能多地去除漂白剂(图2A(i))。用水冲洗胚胎,将C形折叠带移到第一滴水中,使其浸泡30秒,然后将其移到干燥的C形折叠上的另一个区域(图2A(ii))。用水重复冲洗最后一次,让C形折叠放置在干燥C形折叠的未使用部分上一分钟,使其尽可能干燥(图2A(iii))。
8. 同时,将柱塞插入2号玻璃毛细管中,吸干37°C下熔化的1%低熔点琼脂糖VII-A(Sigma),使其刚好位于图2B所示的黑线上方。将毛细管放置在倒置的培养皿上,让琼脂糖在毛细管中冷却一分钟。
9. 用解剖针从C形皱襞中取出干燥的胚胎,并将其置于毛细血管的中心(图2B)。
10.  用细铜丝将胚胎推入凝固琼脂糖的中心,深度约为0.5 cm,并使其与毛细管平行(图2C)。一旦胚胎处于所需位置,将铜线从琼脂糖中缓慢取出。
等待5分钟,让琼脂糖凝固。
注意:将胚胎对准毛细血管的中心,确保其牢固地固定在琼脂糖中。如果它粘在玻璃毛细管上,将其推回琼脂糖中,以避免成像时滑落。这一步需要很大的耐心和练习。这是定位胚胎以获得最高分辨率图像的最佳方法。1%低熔点琼脂糖的等分份可保留在37°C,长期使用或需要多次冷却熔化。但是,在37°C下长时间或经过多次热-冷循环后,它最终将停止凝固。这要求丢弃正在使用的小份,然后使用新的小份。
11.  打开Light sheet Z.1系统并启动Zen 2014 SP软件。将高压灭菌水注入样品室,使其充满制造商协议中所示的边缘。
12.  毛细管中的琼脂糖凝固后,根据制造商手册将毛细管装入样品架中,并拧紧螺钉将其固定。将样品架装入灯片Z.1中,并将其放入装满高压灭菌水的样品室中。慢慢地将胚胎从毛细血管中推出,使其刚好位于毛细血管外并与物镜对准(图2D)。
13.  使用光片Z.1 10x/0.2照明光学元件旋转毛细管,使样品的腹侧表面(v)朝向物镜,以获得腹侧表面的高分辨率图像(图2D)。使用光板Z.1照明物镜40x/1.0(浸水)获取图像。设置功率为13%的20mw 561nm激光器,曝光时间为12.5ms,功率为10%的30mw488nm,曝光时间为29.95ms。以1μm的间隔设置z堆栈,并将z堆栈顶部表示为胚胎顶部上方约20个部分(20μm),z堆栈底部约为
胚胎底部20个切片(20μm)。这是为了确保在成像过程中胚胎移位时数据不会丢失。在处理过程中,可以移除多余的z-stack部分。每2.5分钟在两个通道中设置一个带有双面照明的时间序列采集,持续500个周期。对于不暴露在蓝光下的胚胎,仅使用561nm激光。让图像采集运行大约22小时。
14.  成像完成后,向上拉动柱塞,将胚胎重新插入毛细管,并从光片Z.1上取下样品架。用琼脂糖将胚胎排出并丢弃。毛细管可重复使用多次。
 
F、 光片显微术对神经元损伤的成像
1. 一个定制的开放式光片显微镜被用来获取斑马鱼幼虫的视频。
2. 简单地说,将样品安装在定制的光片显微镜室中,放置在水平台上,由超声电动10nm步进Z驱动控制。
3. 使用样品安装台前面的单个照明臂生成一个光片,通过10x照明物镜传送激光束(488 nm或561 nm)。
4. 通过超高NA(1.0;XLUMPLFN20XW)20倍水浸镜头,在滨松Orca Flash 4.0 V3 sCMOS相机上采集图像。
5. 在带有流选项的变形软件中使用多维采集来获取数据。
6. 使用采集协议以每分钟2μm步长采集1个z堆栈,持续30分钟。
7. 通过德州红/mCherry/Alexafluor594二向色滤过的561nm激光照射样品。在第5分钟和第6分钟,用X-Cite 120LED系统发出的高强度透射白光照射舞台45秒。在接下来的25分钟内恢复延时成像。
8. 获得第一个延时后,再次重复整个过程。
9. 从延时图像制作视频。
 
G、 线粒体代谢通量测定达尼奥雷里奥
1. 将注射的胚胎(C)分成两个培养皿中48h,一个用于光照条件,另一个作为黑暗对照。
2. 用最少的反射光反射两个盘子。
3. 将实验胚胎暴露在立体镜上的高强度LED灯下2小时。然后用细巴斯德吸管将胚胎装入96孔板中。
4. 使用XF96细胞外流量分析仪测量耗氧量(OCR)。
5. 将一个胚胎(48hpf)放入175μl E3水中,开始实验。
6. 每8分钟测量一次OCR,持续6个周期(48分钟),然后添加药物。
7. 根据每种药物的储备浓度计算所需的注射量,以确保井中的最终浓度为-a)9.4 M寡霉素,b)2.5μM FCCP和c)20 mM叠氮化钠。
8. 实验总结如图3所示。
 
 
图3。瞬时表达Aβ-CRY2-mCherry胚胎代谢通量测定示意图
 
H、 三磷酸腺苷(ATP)测定斑马鱼
1. 用(程序C)中的剩余胚胎进行ATP测定。或者重复程序C以产生更多的胚胎。
2. 将5个48h的胚胎置于1.5ml的微管中,加入50μl的10%TCA冷冻缓冲液。对于每种情况,至少要有8个这样的池(n=8)。
3. 用一个手持的小杵均质器,在冰上使胚胎均匀化。
4. 将匀浆在4°C、10000 RPM下离心5分钟。
5. 将上清液转移到新试管中,并保持-80°C以备以后使用,或立即使用。
6. 将5μl样品上清液或ATP标准液加入到白色96孔微量滴定板中(图4)。
7. 使用BioTek cytation 3平板阅读器测量ATP发光,预编程后注入亚砷酸盐ATP缓冲液(150μl/孔),然后注入萤火虫灯笼提取物(45μl/孔)。
 
 
图4。示意图显示了使用斑马鱼的程序摘要
 
一、 荧光显微镜观察线虫Aβ聚集体
1. 把板上的线虫清洗到Eppendorf管中。
2. 在蜗杆悬浮液中加入1毫米左旋咪唑。
3. 轻轻混合蠕虫悬浮液,并将20μl蠕虫悬浮液置于含有2%琼脂糖的玻片上[见Verbruggh和Chan(2011)关于如何制作琼脂糖垫]。
4. 关闭环境光,带上幻灯片,使用561nm和488nm激光进行成像。
5. 用488nm激光,筛选GFP阳性的咽部线虫作为转基因动物。
6. 用561nm激光,观察AΒ的表达和聚集水平。
 
J、 用于确认线虫聚集诱导缺陷的其他试验
1. 寿命测定(He,2011)、线粒体代谢通量测定(Ng和Gruber,2019)和ATP测定(Schaffer等人,2011)可用于确认聚集诱导的缺陷。
 
数据分析
 
A、 光片显微术在胚胎发生过程中对神经发育的成像
1. 在ZEN 2014 SP软件中处理采集的图像文件。
2. 在成像过程中,胚胎可能会轻微移动。因此,确定在每个时间点(ZT)包括胚胎顶部的z-stack截面(图5)。此后,注意胚胎中的z-stack部分(ZB)在失去焦点的整个时间点保持聚焦(图5显示了控制胚胎的顶部、底部和中间z-stack图像的示例,在原始研究论文[Lim等人,2020]中,这些图像用于生成视频3。创建一个子集文件,包括从ZT到ZB的图像。
3. 如果胚胎在某个时间点(T1)后没有显示出任何发育变化,则从成像开始到T1再创建一个子集。
4. 创建最终子集文件的最大强度投影,并调整最适合图像的显示设置。以所选格式将时间序列导出为电影。
 
 
图5。在数据文件的后处理过程中,对胚胎的顶部(ZT)和底部(ZB)z堆栈进行注释,以创建子集文件。ZT被定义为胚胎在每个时间点的顶部(ZT),ZB是z堆栈,在该z堆栈处,胚胎在整个时间点聚焦,之后焦点丢失。比例尺显示50μm。
 
B、 线粒体代谢通量测定(图4)斑马鱼
1. 使用海马波浪控制器软件,计算以下曲线下面积(AUC):
AUC1=氧气消耗率测量值1到6(注射前)
AUC2=耗氧率测量值7到12(注射寡霉素后)
AUC3=耗氧率测量值13到18(FCCP注射后)AUC4=耗氧率测量值19到24(叠氮化钠注射后)
2. 测定相应的呼吸速率,如下所示:
基础呼吸=AUC1–AUC4
质子泄漏=AUC2–AUC4
ATP相关呼吸=AUC1–AUC2
最大呼吸=AUC3–AUC4
备用呼吸能力=AUC3–AUC1
非线粒体呼吸=AUC1–AUC4
3. 用柱状图绘制个体呼吸频率,用单因素方差分析法分析群体平均值。
 
C、 ATP测定斑马鱼
1. 使用根据ATP标准构建的标准曲线计算所有样本的ATP水平。
2. 计算不同条件下的平均ATP水平,并用单因素方差分析对数据进行分析。
 
笔记
 
对于D.melanogaster光片Z.1中的样品成像,由于2.5分钟的短时间间隔,样品室显著升温。因此,将室温设置为18°C,以防止室中的水过热,从而阻止胚胎发育。或者,可以通过在样品室上安装一个蠕动泵来冷却水。
 
食谱
 
1. 标准飞行食品
6g巴氏琼脂
114克葡萄糖
56克玉米粉
25g啤酒酵母
20毫升10%尼泊金,每升1升
2. 葡萄汁琼脂平板
使用FlyStuff葡萄琼脂预混料制备葡萄汁琼脂,用于胚胎收集(25
根据制造商的说明,并在每个60mm培养皿中倒入约4-5ml琼脂。使板在4°C下固化和储存。
3. 酵母糊
将酵母(布鲁格曼速溶干酵母蓝,500克)与少量的水混合在一个塑料烧杯中,以获得粘稠的糊状物。酵母糊可保存在4°C下覆盖铝箔的烧杯中。
4. 1%低胶凝温度琼脂糖于1.5毫升的Eppendorf试管中
a、 在50 ml微波安全玻璃中,添加0.1 g琼脂糖,低凝胶温度VII-a型,并添加10 ml高压灭菌蒸馏水
b、 微波炉间歇搅拌,直到琼脂糖在没有煮沸的情况下溶解,以防止水分过度流失
c、 一旦琼脂糖完全溶解,让混合物稍微冷却,然后将大约1毫升1%的琼脂糖等分到1.5毫升的Eppendorf试管中
d、 琼脂糖凝固后关闭Eppendorf试管,并在室温下保存
e、 安装时,将1%低胶凝温度的琼脂糖在70°C下在加热块中熔化半小时,然后冷却至37°C,在使用安装前通过倒置充分混合
5. 2%低胶凝温度琼脂糖溶于1.5 ml Eppendorf试管中(用于秀丽隐杆线虫成像)
同上,但将0.2 g琼脂糖、低凝胶温度VII-A型溶解在10 ml高压灭菌蒸馏水中
6. 60x E3(胚胎培养基)储备液
a、 将172 g NaCl、7.6 g KCl、29 g CaCl2·2H2O和49 g MgSO4·7H2O溶解在10 L Milli-Q水中
b、 在蒸馏水中稀释160 ml 60x储备溶液,以形成10 L E3(1x)
7. 2%低熔点琼脂糖
a、 将1g琼脂糖粉溶于100ml E3(1x)
b、 在微波炉中以低功率烹饪2分钟(用于鱼类成像)
8. 25x三胺原液(用于D.rerio)
a. Dissolving 400 mg Tricaine in 97.9 ml DD water
b. Adjust pH to 7 using Tris-HCl (1 M)
c. To use Tricaine for anesthesia, dilute stock solution by adding 96 ml E3 (1x) to 4 ml of 25x stock solution in a beaker
d. Diluted Tricaine should be disposed in activated charcoal
9. 10% TCA buffer
Dissolve 1 g TCA powder in 10 ml distilled water
10.  Arsenite ATP buffer Mix 80 mM MgSO4·7H2O
10 mM KH2PO4
100 mM Na2HASO4·7H2O in 1:1:1 ratio
 
Acknowledgments
 
We are thankful for the funding provided by Ministry of Education Singapore AcRF grant IG17LR005, IG17-BS101 and IG18-BS002 to JG, Yale-NUS College grant R-607-265-225-121 to ASM, AcRF grants IG17-LR006 and IG18-LR001 to NST.
 
Competing interestsCompeting interests
 
We have no conflicts of interest to declare.
 
Ethics
 
Institutional biosafety and genetic manipulation guidelines of IMCB were followed for generation of transgenic zebrafish. Fish husbandry, rearing and maintenance were performed following approved protocols by Institutional Animal Care and Use Committee (IACUC) of the Biological Resource Center at A*STAR. Approved experimental protocols (IACUC 191501) were followed.
 
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Copyright Kaur et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kaur, P., Kibat, C., Teo, E., Gruber, J., Mathuru, A. and Tolwinski, N. (2020). Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans. Bio-protocol 10(23): e3856. DOI: 10.21769/BioProtoc.3856.
  2. Lim, C. H., Kaur, P., Teo, E., Lam, V. Y. M., Zhu, F., Kibat, C., Gruber, J., Mathuru, A. S. and Tolwinski, N. S. (2020). Application of optogenetic Amyloid-beta distinguishes between metabolic and physical damages in neurodegeneration. Elife 9: e52589.
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