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Oct 2018

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A Parkinson’s Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells
LUHMES细胞中与帕金森氏病相关的线粒体和神经元形态学高通量筛选分析   

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Abstract

Parkinson’s disease is a devastating neurodegenerative disorder affecting 2-3% of the population over 65 years of age. There is currently no disease-modifying treatment. One of the predominant pathological features of Parkinson’s disease is mitochondrial dysfunction, and much work has aimed to identify therapeutic compounds which can restore the disrupted mitochondrial physiology. However, modelling mitochondrial dysfunction in a disease-relevant model, suitable for screening large compound libraries for ameliorative effects, represents a considerable challenge. Primary patient derived cells, SHSY-5Y cells and in vivo models of Parkinson’s disease have been utilized extensively to study the contribution of mitochondrial dysfunction in Parkinson’s. Indeed many studies have utilized LUHMES cells to study Parkinson’s disease, however LUHMES cells have not been used as a compound screening model for PD-associated mitochondrial dysfunction previously, despite possessing several advantages compared to other frequently used models, such as rapid differentiation and high uniformity (e.g., in contrast to iPSC-derived neurons), and relevant physiology as human mesencephalic tissue capable of differentiating into dopaminergic-like neurons that highly express characteristic markers. After previously generating GFP+-LUHMES cells to model metabolic dysfunction, we report this protocol using GFP+-LUHMES cells for high-throughput compound screening in a restoration model of PD-associated mitochondrial dysfunction. This protocol describes the use of a robust and reproducible toxin-induced GFP+-LUHMES cell model for high throughput compound screening by assessing a range of mitochondrial and neuronal morphological parameters. We also provide detailed instructions for data and statistical analysis, including example calculations of Z’-score to assess statistical effect size across independent experiments.

Keywords: Parkinson’s disease (帕金森氏病), Mitochondria (线粒体), Compound screening (化合物筛选), LUHMES (LUHMES)

Background

Parkinson’s disease (PD) is a neurodegenerative disorder characterised primarily by loss of dopaminergic neurons in the substantia nigra of the midbrain and the accumulation of α-synuclein in intra-neuronal inclusions. It is the second most common neurodegenerative disorder affecting 2-3% of the population over 65 years of age (Poewe et al., 2017). Patients present with resting tremors, bradykinesia and muscle rigidity, along with non-motor symptoms which can include depression, anosmia and memory problems. There is currently no disease-modifying treatment that can prevent or slow the progression of PD, presenting an urgent need for an effective therapeutic (Armstrong and Okun, 2020).

Mitochondrial dysfunction is a key pathological hallmark of PD; many of the genetic loci associated with familial PD encode proteins that are involved in mitochondrial function or regulation (Hauser and Hastings, 2013). A variety of mitochondrial pathways are disturbed in PD pathology including ATP production, mitophagy, trafficking, biogenesis and calcium buffering (Park et al., 2018). It is therefore unsurprising that mitochondrial dysfunction is a popular target for therapeutic discovery in PD, which aims to identify compounds capable of enhancing those mitochondrial pathways disturbed in the disease state back towards normal physiological levels. This presents the challenge of finding an effective method to model mitochondrial dysfunction in vitro whereby large compound libraries can be screened efficiently within a disease-relevant model.

We have previously reported the use of primary patient fibroblasts to screen for compounds which rescue mitochondrial phenotypes seen in those fibroblasts (Mortiboys et al., 2013). Furthermore, recently a patient derived iPSC neuron model using a high-throughput, semi-automatic, imaging system was described to identify compounds which ameliorate mitochondrial clearance deficits (Yamaguchi et al., 2020). Both of these systems utilize patient derived cells, one potential issue when using patient derived models is the amount of material required and uniformity of sample. An alternative approach is the use of Lund human mesencephalic (LUHMES) cells. LUHMES cells are a subclone of the MESC2.10 cell line, derived from human embryonic ventral mesencephalic tissue and immortalised by integration of a v-myc retroviral factor which is tetracycline regulatable (Lotharius et al., 2002 and 2005). Briefly, LUHMES cells exist in a proliferative state until the addition of tetracycline, dibutyryl cyclic AMP (dCAMP) and glial cell derived neurotrophic factor (GDNF) which halts proliferation and induces uniform differentiation into a dopaminergic-like phenotype. As PD predominantly affects the dopaminergic neurons of the substantia nigra, the differentiated LUHMES cell phenotype is highly physiologically relevant as a model. Further characterisation has demonstrated that differentiated LUHMES cells resemble primary neuron cultures in many aspects: broad upregulation of neuronal markers, extensive neurite outgrowth and basic electrophysiological features (Scholz et al., 2011). Unlike many other transformed neuronal cell lines, LUHMES cells in the differentiated state have c-myc switched off and no dysregulation of the cell cycle is seen. This is particularly important when considering cell cycle mechanisms involved in neuronal DNA repair and neurodegeneration modelling. LUHMES cells are candidates for use in high-throughput screening due to their ease of handling, uniformity and high purity following differentiation induction (> 99%) compared to the often capricious and phenotypically variable culturing of primary neurons (Scholz et al., 2011). A comparative study also showed LUHMES cells express higher levels of neuronal markers (TUBB3, ENO2, MAP2) and have increased neurotoxicant sensitivity compared to two other commonly used neuronal models, SH-SY5Y neuroblastoma cells and human foetal neural stem cells (Tong et al., 2017).

LUHMES cells have been used extensively in high-throughput screening for the identification of neurotoxicants (Stiegler et al., 2011; Krug et al., 2013), including one specific to mitochondrial toxicity (Delp et al., 2019). However, we have only found one study to date that has exploited the high-throughput potential of LUHMES cells in PD therapeutic discovery. Höllerhage et al. used a previously developed alpha-synuclein LUHMES model to screen 1,600 FDA-approved drugs for protective effects by measuring cell viability after treatment (Höllerhage et al., 2017). Previously, we reported the generation of GFP+-LUHMES and the use of them to model metabolic dysfunction in co-culture with astrocytes (Ratcliffe et al., 2018).

Here we utilise the GFP+-LUHMES which we previously generated and validated as a useful model for metabolic dysfunction, to present a detailed and reproducible protocol for high-throughput screening of compounds in a toxin-induced differentiated LUHMES cell model relevant to PD. This protocol improves on previous studies by using high-content live cell imaging and analysis to assess mitochondrial parameters and neuronal morphology.

In the approach described here, differentiated LUHMES cells are treated with rotenone to model PD-associated mitochondrial dysfunction and neuronal loss in accordance with previous studies (Krug et al., 2013; Dolga et al., 2014). LUHMES cells are grown to full confluency before the addition of differentiation factors, and then replated into 384-well plates after two days. The cells remain in differentiation media for the full time course of the experiment. The plate is treated with rotenone on the fifth day, then treated with test compounds on the sixth day and live imaged on the seventh day (Figure 1). When designing a toxin induced model for screening; there are generally two paradigms. The first being a protection model, where the potential beneficial compound is added to the cells before the toxin. Secondly, and is the case in this protocol, is a restoration model; where the toxin is added first and then the potential beneficial compounds are added afterwards. This is testing the beneficial compounds to restore a defect already present as opposed to protecting from damage occurring. The advantage of this technique lies in the relative ease and speed in generating a uniform population of disease-relevant human dopaminergic neurons that can be used for large-scale compound screening. Furthermore, high-content live cell imaging and analysis allows the assessment of compound dose-response effects on a broad range of mitochondrial and neuronal morphological parameters.


Figure 1. Timeline of the GFP-LUHMES drug screening protocol

Materials and Reagents

Materials

  1. 384-well black plates (Greiner-Bio, catalog number: 781091)

  2. 384-well LDV source plates (Labcyte, catalog number: LP-0200)

  3. 384-well source plate seals (Fluidx, catalog number: 41-1011)

  4. 5 ml serological pipettes (Fisher Scientific, catalog number: 13-676-10H)

  5. 10 ml serological pipettes (Fisher Scientific, catalog number: 13-676-10J)

  6. 25 ml serological pipettes (Fisher Scientific, catalog number: 13-678-11)

  7. 10 μl pipette tips (Fisher Scientific, catalog number: 02-707-441)

  8. 200 μl pipette tips (Fisher Scientific, catalog number: 02-707-422)

  9. 1,000 μl pipette tips (Fisher Scientific, catalog number: 02-707-402)

  10. 15 ml Falcon tubes (Greiner-Bio, catalog number: 188271)

  11. 50 ml Falcon tubes (Greiner-Bio, catalog number: 227261)

  12. T75 flasks (Greiner-Bio, catalog number: 658175)

  13. Cryovials (Greiner-Bio, catalog number: 122261)


    Reagents

  1. LUHMES cells (ATCC® CRL-2927TM)

  2. GFP-LUHMES cells (RRID: CVCL_B056, Ratcliffe et al., 2018)

  3. GFP-expressing lentiviral particles

  4. Advanced DMEM/F-12 (Thermo Scientific, catalog number: 12634010)

  5. Fibronectin (Sigma-Aldrich, catalog number: FC010)

  6. Poly-L-Ornithine (Sigma-Aldrich, catalog number: P4957)

  7. PBS Tablets (Thermo Scientific, catalog number: BR0014G)

  8. Trypsin 10x (Lonza, catalog number: BE02-007E)

  9. L-Glutamine (Lonza, catalog number: BE-17-605E)

  10. N-2 Supplement (Gibco, catalog number: 11520536)

  11. Pen-Strep (Lonza, catalog number: DE17-603E)

  12. FGF-basic (Peprotec, catalog number: 100-18B)

  13. dCAMP (Sigma-Aldrich, catalog number: D0627)

  14. GDNF (Peprotech, catalog number: 450-10)

  15. Tetracycline (Sigma-Aldrich, catalog number: T7660)

  16. Hoechst (Sigma, catalog number: 94403)

  17. TMRM (Invitrogen, catalog number: T668)

  18. Sterile 1x PBS (see Recipes)

  19. Sterile 1x Trypsin (see Recipes)

  20. LUHMES Base Media (see Recipes)

    Advanced DMEM/F-12

    N-2 Supplement

    Pen-Strep

    L-Glutamine

  21. LUHMES Proliferation Media (see Recipes)

    LUHMES Base Media

    FGF-basic

    LUHMES Differentiation Media

    Tetracycline

    dCAMP

    GDNF

Equipment

  1. Mechanical pipette gun for serological pipettes

  2. Mechanical pipettes (P20, P200, P1000)

  3. Multichannel mechanical pipettes (P10, P200)

  4. 4 °C fridge

  5. -20 °C freezer

  6. -80 °C freezer

  7. Sterile tissue culture hood

  8. Autoclave

  9. Centrifuge for tissue culture (MSE Harrier 15/80, catalog number: MSB080.CX1.5)

  10. Centrifuge for source plates

  11. CO2 Incubator (Sanyo, model: MCO-19AIC)

  12. Echo 550 Liquid Handler (Labcyte, catalog number: Echo 550)

  13. MultiPod Controller (Roylan Developments, catalog number: SPOD0012)

  14. StoragePod Enclosure (Roylan Developments, catalog number: SPOD0010)

  15. Opera Phenix High-Content Screening System (PerkinElmer)

Software

  1. Echo Liquid Handler Software (Labcyte)

  2. Echo Plate Reformat (Labcyte)

  3. Harmony High-Content Imaging and Analysis Software (PerkinElmer)

  4. Columbus Image Data Storage and Analysis System (PerkinElmer)

  5. Excel 2016 (Microsoft)

  6. GraphPad Prism 8.2 (GraphPad)

Procedure

Generation of GFP-LUHMES cells

GFP-LUHMES cells are generated by the transduction of proliferative LUHMES cells with GFP-expressing lentiviral particles under the control of a PGK promoter (Ratcliffe et al., 2018).

  1. Seed proliferative LUHMES cells (p3-p5) at 1.5 x 106 per T75 flask.

  2. Transduce with GFP-expressing lentiviral particles for 24 h. MOI of 8; for transduction, a 1:100 dilution was used. Transduction efficiency was previously assessed by FACS sorting to be 98.5%. GFP-expressing lentiviral particles were a kind gift from Eva Karyka and Mimoun Azzouz at the University of Sheffield.

  3. After 24 h, maintain in LUHMES Proliferation Media for 72 h before beginning experiments.


    Part I: GFP-LUHMES cell Maintenance Protocol


  1. Defrost and seed GFP-LUHMES cells in a sterile culture hood

    1. Prepare LUHMES Proliferation Media and sterile 1x PBS according to recipes below.

    2. Prepare a Fibronectin Poly-L-Ornithine coated T75 flask.

      1. Prepare 10 ml of Fibronectin Poly-L-Ornithine Coating Solution per T75 flask by adding 1 μg/ml Fibronectin and 50 μg/ml Poly-L-Ornithine to sterile distilled water.

      2. Mix the Fibronectin Poly-L-Ornithine Coating Solution and transfer 10 ml to the T75 flask using a 10 ml serological pipette.

      3. Incubate the flask with the Fibronectin Poly-L-Ornithine Coating Solution at 37 °C for at least 3 h or overnight.

      4. Remove the Fibronectin Poly-L-Ornithine Coating Solution from the flask using a 10 ml serological pipette.

      5. Wash the coated surface by transferring 5 ml of sterile 1x PBS to the flask using a 10 ml serological pipette and gently tilting the flask from side to side allowing the PBS to run across the whole surface. The PBS should be left in the flask until immediately before the cells are seeded in the flask to prevent the coating from drying out. The flasks can be prepared up to 1 hour before use.

    3. Defrost and seed GFP-LUHMES cells into LUHMES Proliferation Media.

      1. Remove prepared LUHMES Proliferation Media from the fridge and allow to warm to room temperature.

      2. Take one frozen vial of GFP-LUHMES cells from freezer storage. Generally, we recommend to revive a vial of passage 1-10 so that revived cells can undergo numerous splits before retiring at passage 15.

      3. Add 10 ml room temperature LUHMES Proliferation Media to a 15 ml Falcon using a 10 ml serological pipette.

      4. Transfer ~500 μl of room temperature LUHMES Proliferation Media to the GFP-LUHMES cell vial using a P1000 mechanical pipette and slowly mix by aspirating and expelling the liquid in the tip repeatedly.

      5. When the solution in the GFP-LUHMES cell vial is fully defrosted, transfer to the solution to the room temperature LUHMES Proliferation Media in the 15 ml Falcon.

      6. Remove all 1x PBS from the coated T75 flask and add all of the cell solution from the 15 ml Falcon to the T75 flask using a 10 ml serological pipette.

      7. Tilt the flask back and forward and then side to side 2-3 times to distribute the GFP-LUHMES cells evenly over the coated surface.

      8. Incubate the flask in a 37 °C/5% CO2 incubator.

    4. On the following day, change the GFP-LUHMES cell media to remove any cells not fully attached.

      1. Remove all media from the T75 flask using a 10 ml serological pipette.

      2. Add 10 ml room temperature LUHMES Proliferation Media to the T75 flask using a 10 ml serological pipette.

      3. Incubate the flask in a 37 °C/5% CO2 incubator.


  2. Split the GFP-LUHMES cells every 3-4 days or when confluency is 80-90% in a sterile culture hood

    1. Prepare two Fibronectin Poly-L-Ornithine coated T75 flasks according to the procedure described above (Step A2).

    2. Prepare LUHMES Proliferation Media and 1x Trypsin according to the recipes below. If pre-prepared, remove from the fridge and allow to warm to room temperature.

    3. Split GFP-LUHMES cells into two flasks containing LUHMES Proliferation Media. One flask will be used for stock maintenance and one flask will be used for differentiation.

      1. Remove all LUHMES Proliferation Media from GFP-LUHMES cell flask using a 10 ml serological pipette and transfer 5 ml into a 15 ml Falcon.

      2. Add 5 ml room temperature 1x Trypsin to GFP-LUHMES cell flask using a 10 ml serological pipette.

      3. Incubate GFP-LUHMES cell flask for 5 min in a 37 °C/5% CO2 incubator.

      4. Firmly smack the side of the GFP-LUHMES cell flask 2-3 times to dislodge cells into the solution. If successful, cell clouds should be visible in the solution.

      5. Transfer all cell solution from GFP-LUHMES cell flask into the 15 ml Falcon containing 5 ml of used LUHMES Proliferation Media.

      6. On the bench, centrifuge the 15 ml Falcon at 400 x g for 4 min.

      7. Remove the supernatant using a 10 ml serological pipette and discard.

      8. Resuspend the pelleted GFP-LUHMES cells in an appropriate amount of LUHMES Proliferation Media according to desired splitting ratio. We recommend a 1:15 splitting ratio of a fully confluent flask to be ready to split again in 3 days, although this may need to be adjusted slightly depending on cell behavior.

      9. Remove all 1x PBS from the coated T75 flasks and add 10 ml LUHMES Proliferation Media per flask using a 10 ml serological pipette.

      10. Add an appropriate amount of re-suspended GFP-LUHMES cell solution to the LUHMES Proliferation Media in both T75 flasks.


  3. Change the Proliferation Media on the GFP-LUHMES cells every 2 days (following a split)

    1. Remove a Falcon tube of LUHMES Proliferation Media from the fridge and allow to warm to room temperature.

    2. Change the media on the GFP-LUHMES cells to fresh Proliferation Media.

      1. Remove all LUHMES Proliferation Media from GFP-LUHMES cell flask using a 10 ml serological pipette and discard.

      2. Add 10 ml of room temperature LUHMES Proliferation Media to the GFP-LUHMES cell flask using a 10 ml serological pipette.

      3. Incubate the flask in a 37 °C/5% CO2 incubator.


        Part II: GFP-LUHMES cell Differentiation and Replating Protocol


  1. Initiate differentiation of GFP-LUHMES cells (Day 1)

    1. Wait 2-3 days following a split of the GFP-LUHMES cells to allow proliferation.

    2. Prepare LUHMES Differentiation Media according to the recipe below. This must be made fresh before every use.

    3. Change the Proliferation Media on the GFP-LUHMES cells to Differentiation Media.

      1. Remove all LUHMES Proliferation Media from GFP-LUHMES cell flask using a 10 ml serological pipette and discard.

      2. Add 10 ml of room temperature LUHMES Differentiation Media to the GFP-LUHMES cell flask using a 10 ml serological pipette.

      3. Incubate the flask in a 37 °C/5% CO2 incubator.


  2. Replate differentiating GFP-LUHMES into a 384-well plate (catalog number: 781091) (Day 3)

    1. Prepare a Fibronectin Poly-L-Ornithine coated 384-well plate.

      1. Prepare 15 ml of Fibronectin Poly-L-Ornithine Coating Solution per 384-well plate by adding 1 μg/ml Fibronectin and 50 μg/ml Poly-L-Ornithine to sterile distilled water.

      2. Mix the Fibronectin Poly-L-Ornithine Coating Solution and transfer 15 ml to a suitable reservoir for multichannel pipetting (for example but not limited to an upturned pipette box lid, clean and sterile).

      3. Transfer 50 μl of the Fibronectin Poly-L-Ornithine Coating Solution to each well of the 384-well plate except for the two outermost columns and rows using a P200 multichannel mechanical pipette.

      4. Transfer 40 μl of sterile 1x PBS to each of the empty outermost wells to act as a firewall using a P200 multichannel mechanical pipette.

      5. Incubate the flask with the Fibronectin Poly-L-Ornithine Coating Solution at 37 °C for at least 3 h or overnight.

      6. Remove the Fibronectin Poly-L-Ornithine Coating Solution from the 384-well plate using a P200 multichannel mechanical pipette.

      7. Wash the coated surface by transferring 50 μl of sterile PBS to each coated well using a P200 multichannel mechanical pipette.

    2. Prepare LUHMES Differentiation Media according to the recipe below.

    3. Lift GFP-LUHMES cells and seed 10,000 cells/well into a coated 384-well plate.

      1. Remove all LUHMES Differentiation Media from the differentiating GFP-LUHMES cell flask using a 10 ml serological pipette and transfer 5 ml into a 15 ml Falcon.

      2. Add 5 ml room temperature 1x Trypsin to the differentiating GFP-LUHMES cell flask using a 10 ml serological pipette.

      3. Incubate differentiating GFP-LUHMES cell flask for 5 min in a 37 °C/5% CO2 incubator.

      4. Firmly smack the side of the differentiating GFP-LUHMES cell flask 2-3 times to dislodge cells into the solution.

      5. Transfer all cell solution from differentiating GFP-LUHMES cell flask into the 15 ml Falcon containing 5 ml of used LUHMES Differentiation Media.

      6. On the bench, centrifuge the 15 ml Falcon at 400 x g for 4 min.

      7. Remove the supernatant using a 10 ml serological pipette and discard.

      8. Resuspend the pelleted GFP-LUHMES cells in 5 ml of LUHMES Differentiation Media, homogenising thoroughly by aspirating and expelling the GFP-LUHMES resuspension 10-15 times with a P1000 mechanical pipette.

      9. Prepare a Haemocytometer and transfer 10 μl of GFP-LUHMES resuspension onto the counting grid.

      10. Count the number of GFP-LUHMES cells and calculate the cells/ml in the GFP-LUHMES resuspension.

      11. Remove quantity of GFP-LUHMES resuspension containing 4 x 106 cells and transfer to a 15 ml Falcon. This will be the plating suspension.

      12. Top up the plating suspension to a final volume of 12 ml with LUHMES Differentiation Media using a 10 ml serological pipette.

      13. Remove the PBS from the coated wells on the first half of the 384-well plate using a P200 multichannel mechanical pipette.

      14. Homogenise the plating suspension thoroughly by aspirating and expelling the solution 10-15 times with a 10 ml serological pipette.

      15. Transfer half of the plating suspension (6 ml) into a suitable reservoir for multichannel pipetting.

      16. Transfer 30 μl of plating suspension to each coated well of the 384-well plate.

      17. Remove the PBS from the coated wells on the second half of the 384-well plate using a P200 multichannel mechanical pipette.

      18. Re-homogenise the plating suspension in the 15 ml Falcon thoroughly by aspirating and expelling the solution 10-15 times with a 10 ml serological pipette.

      19. Transfer the rest of the plating suspension to the reservoir and fill the remaining coated wells with 30 μl of plating suspension per well using a P200 multichannel mechanical pipette.

      20. Move the plate back and forth, then side to side, several times to ensure the cells are evenly distributed across the well bottoms.

      21. Incubate the 384-well plate in a 37 °C/5% CO2 incubator.


        Part III: GFP-LUHMES Drug Screening Protocol (timeline continued from Part II)


  1. Treat differentiated GFP-LUHMES 384-well plate with rotenone (Day 5)

    1. Prepare LUHMES Differentiation Media according to the recipe below.

    2. Prepare rotenone-treated media and vehicle-treated media in a sterile culture hood.

      1. To avoid cell disturbance, we avoid removing media and instead top up each well with 10 μl rotenone-treated media giving a final volume of 40 μl media per well. Therefore, rotenone-treated media must be prepared at four times the desired final concentration (4 μM).

      2. Transfer 4 ml of LUHMES Differentiation Media to a 15 ml Falcon and label “Rotenone”. Transfer 4 ml of LUHMES Differentiation Media to another 15 ml Falcon and label “Vehicle”.

      3. Prepare 10 mM rotenone in ethanol. Rotenone powder should be weighed in a fume hood.

      4. Add 6.4 μl 10 mM rotenone to the media in the 15 ml Falcon labeled “Rotenone” using a P20 mechanical pipette. The final rotenone concentration in the well will be 4 μM.

      5. Add an equal volume of ethanol (6.4 μl) to the 15 ml Falcon labeled “Vehicle” using a P20 mechanical pipette.

      6. Mix both 15 ml Falcons by slowly inverting 3-4 times.

    3. Transfer treated media to the differentiated GFP-LUHMES 384-well plate. We suggest a plate map as shown in Figure 2A (i.e., “DMSO vehicle” wells are treated with vehicle-treated media, and all other wells treated with rotenone-treated media).

      1. Transfer the contents of “Rotenone” media Falcon and “Vehicle” media Falcon into separate reservoirs for multichannel pipetting. Label the reservoirs accordingly.

      2. Transfer 10 μl of rotenone-treated media to each well of the 384-well plate using a P10 multichannel mechanical pipette, ensuring the pipette tips are placed deep enough in the well to ensure ejected liquid mixes with the contents of the well. Leave some wells untreated for addition of vehicle-treated media to measure rotenone effect later. We suggest including vehicle-treating some wells in each DMSO only control column as explained in Step B3 below. Transfer 10 μl of vehicle-treated media to each untreated well of the 384-well plate using a P10 multichannel mechanical pipette.

      3. Incubate the 384-well plate in a 37 °C/5% CO2 incubator.


  2. Prepare compounds in DMSO and write protocols for using the Echo 550 Liquid Handler and Opera Phenix High-Content Screening System

    1. Prepare compound library in 100% anhydrous DMSO on 384-well source plates (catalog number: LP-0200) with plate seal. Store in the StoragePod Enclosure and maintain a dry nitrogen atmosphere using the MultiPod Controller.

    2. Write a compound transfer protocol using the Echo Liquid Handler software. The source plate should be set to “384LDV-DMSO”, the destination plate to “Griener_384PS_781096” and custom mapping should be selected.

    3. Set the protocol to transfer an appropriate volume from each well of the source plate to the corresponding well on the destination plate to give the desired final drug concentration. We suggest splitting the destination plate into quarters and using the first column of each quarter for DMSO only treatment to act as a negative control (Figure 2). Parameter values for each compound-treated well in each quarter can then be normalised to the respective DMSO control well values in that quarter. For a full dose response curve, we recommend testing 8 concentrations for each compound at every half-log, i.e., 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1,000 nM, 3,000 nM, 10,000 nM. We also recommend including at least one positive control compound on each plate; we used a compound previously identified in house that showed beneficial effects on mitochondrial and morphological parameters.



      Figure 2. Plate map and example images following assaying of a GFP-LUHMES 384-well plate drug screen. A. Plate map showing the suggested arrangement of control wells and test compound wells. All wells are treated with rotenone except for “DMSO vehicle” wells. The colours represent the concentration in nM for each compound. B-D. Blue = DAPI, orange = TMRM, green = GFP, scale bars = 20 μm. Z stack maximum projection confocal images captured using the Opera Phenix High-Content Screening System from (B) a vehicle-treated DMSO control well, (C) a rotenone-treated DMSO control well and (D) a rotenone-treated positive control well (1,000 nM).


    4. Write an imaging protocol (details below) for imaging the LUHMES cells on the Harmony High-Content Imaging and Analysis Software. Use the DAPI, Alexa 488 and Cy3 channels to capture the Hoechst signal, GFP signal and TMRM signal respectively. Use test images to ensure appropriate exposure time for each laser; this needs to be set by the user as it depends on the signal strength of laser power of each imaging system. Use a 40x water confocal objective, set the number of fields to 10 and set the number of stacks to 6. Select the wells to be imaged (note the outermost two columns and rows cannot be imaged by the Opera Phenix) and select the location of the fields in each well.


  3. Treat differentiated GFP-LUHMES 384-well plate with compounds (Day 6)

    1. Depressurise the StoragePod Enclosure using the Multipod Enclosure and remove the 384-well source plate.

    2. Centrifuge the 384-well source plate at 2,500 x g for 2 min to remove any gas bubbles from the plate contents.

    3. Calibrate and focus on the Echo 550 liquid handler before each independent plate shoot.

    4. Survey the 384-well ldv source plate using the Echo 550 liquid handler. Ensure the volume in each well is high enough for transfer of the desired volume to the destination plate according to the compound transfer protocol. Note that the volume of each well in the 384-well source pate should not exceed 12 μl. Ensure that the water content of the contents of each well does not exceed 30% (this is inferred from the measurement of DMSO content which should not be lower than 70%; if this is the case then the contents of that well must be refreshed).

    5. Remove the seal from the 384-well source plate and initiate the compound transfer protocol. Transfer the 384-well source plate and the destination plate into the machine when prompted.

    6. When the protocol is complete, remove the destination plate and incubate immediately in a 37 °C/5% CO2 incubator.

    7. Replace the seal on the 384-well source plate and depressurise the StoragePod Enclosure using the Multipod Controller. Replace the 384-well source plate in the StoragePod Enclosure. Lock and repressurise the StoragePod Enclosure using the MultiPod Controller.


  4. Assay and image the GFP-LUHMES 384-well plate (Day 7)

    1. Prepare LUHMES Differentiation Media according to the recipe below.

    2. Prepare the assay media.

      1. Prepare 10 mM Hoechst in sterile distilled water.

      2. Prepare 80 μM TMRM in DMSO.

      3. Transfer 4 ml of LUHMES Differentiation media into a 15 ml Falcon.

      4. Add 20 μl of x mM Hoechst solution to the 15 ml Falcon.

      5. Add 20 μl of x mM TMRM solution to the 15 ml Falcon.

      6. Mix the contents of the 15 ml Falcon (assay media) by slowly inverting the Falcon 4-5 times.

    3. Incubate the GFP-LUHMES 384-well plate with assay media for 1 h.

      1. Transfer the assay media to a suitable reservoir for multichannel pipetting.

      2. Transfer 10 μl of assay media to each well of the 384-well plate using a P10 multichannel mechanical pipette, ensuring the pipette tips are placed deep enough in the well to ensure ejected liquid mixes with the contents of the well.

      3. Incubate the GFP-LUHMES 384-well plate in a 37 °C/5% CO2 incubator for 1 h.

    4. Image the GFP-LUHMES 384-well plate using the Opera Phenix High-Content Screening System.

      1. Set the TCO (temperature and CO2) settings on the Opera Phenix to 37 °C and 5% CO2 and set both to “On”. Open the connected CO2 gas cylinder when prompted. Allow the system to reach the desired temperature and gas level.

      2. Open the prepared imaging protocol.

      3. Load the GFP-LUHMES 384-well plate.

      4. Flush the water objective lens.

      5. Run a test and adjust the exposure time of each channel so that the intensity is significant over background levels. Adjust the height of the stacks if needed so that images are well focused across the height of the cell.

      6. Run the imaging protocol (Figure 2).

Data analysis

The images will be analysed in batch using the Columbus Image Data Storage and Analysis System. The GFP-signal will be used to assess neuronal morphology parameters and the TMRM-signal will be used to assess mitochondrial function and morphology.

A short video (Video 1) is available to show the analysis software and provide guidance to parts of the protocol, with detailed instructions below.


Video 1. Analysis set up using Columbus analysis software

  1. Once the imaging protocol is completed, export the measurement file to a convenient location. Ensure “Measurements Inc. Associated Files” is selected and note the export path.

  2. Import the exported files to the Columbus Image Data Storage and Analysis System.

  3. Select the Image Analysis tab and open the imported file.

  4. Write an analysis protocol using the following building blocks. Add a new building block by clicking the blue plus symbol.

    1. Input Image. This is where the images are loaded into the analysis tool. Select “Maximum Projection” for Stack Processing and select “Basic” for Flatfield Correction. Uncheck the box next to Quick Tune.

    2. Find Nuclei. This first Find Nuclei building block is used to aid the GFP-neurite detection in the following building blocks. The Hoechst signal will be used later to more accurately count the nuclei. Set the channel to “Alexa 488” (GFP) and use Method A. Label the Output Population as “Nuclei (GFP neurites)”.

    3. Find Neurites. Set the Channel to “Alexa 488" (GFP) and ensure Population is set to “Nuclei (GFP neurites)” and Region to “Nucleus”. Two selections will be made, shown by the tabs above the image. The first is made via the positions of the nuclei selected above, and labeled “Nuclei (GFP neurites)” (Figure 3A). The second detects any neurite structures in the image, labeled “Neurite Segments”. Click the black downward-facing arrow and adjust the values for each detection parameter so that as many neurites are selected as possible. Do not be concerned about background detection in the “Neurite Segments” selection as this will be removed in the following building block. Label the Output Population as “Neurite Segments”.

    4. Select Population. We will now filter out background debris detected in the Find Neurites selection above. Set the Population to “Neurite Segments” and the Method to “Filter by Property”. Expand the drop down menu by clicking the black downward-facing arrow. Select “Segment Length”, “Neurite Segment Area” and “Neurite Segment Roundness” and adjust the values until most of the debris is removed from selection but the ‘real’ neurites remain selected. We used values of > 12.5, > 0.7, and > 0 respectively. Label the Output Population as “Neurite Segments Selected”.

    5. Calculate Morphology Properties. Select Population “Neurite Segments Selected” and use Method “Standard”. Expand the drop down menu and check the box next to each parameter.

    6. Calculate Morphology Properties (2). Select Population “Nuclei (GFP neurites)”, Region “Neurite Tree” and Method “Standard”. Expand the drop down menu and check the box next to each parameter.

    7. Find Image Region. Select Channel “Cy3”, ROI Population to “None” and Method to “Common Threshold”. Expand the drop down menu and change the threshold to an appropriate level to select most of the signal without excessive background. Tick “Split into Objects”, expand the further drop down menu and change the minimum area to an appropriate value to remove background (we used > 25 μm2). Leave “Fill Holes” unticked. Label the Output Population “Image Region (Cy3)”.

    8. Find Image Region (2). Repeat as in (g.) but use Channel “Alexa 488” to select the GFP-neurite image region (Figure 3B). Label the Output Population “Image Region (GFP-neurites)”.

    9. Filter Image. We will now segment the mitochondria. Set Channel “Cy3” and Method to “Texture SER”. Expand the drop down menu and set Filter to “SER Ridge”, Scale to 1 px and Normalization to “Unnormalized”. Label the Output Image as “SER Ridge”.

    10. Calculate Image. This step amplifies the signal to improve segmentation. Set Method to “By Formula” and enter the Formula “A*1000”. Set Channel A to “SER Ridge” and label the Output Image as “Calculated Image”.

    11. Find Spots. Set Channel “Calculate Image” and the ROI Population to “Image Region (GFP-neurites)”. This ensures only mitochondria within the GFP-neurite selection will be segmented. Set Method “D” and label the Output Population as “Mitochondria”.

    12. Select Population. Set Population to “Mitochondria” and Method to “Filter by Property”. Choose the parameter “Spot Area [px2]”, select “>” and enter a minimum value to remove any debris in the selection (we used 8 px2) (Figure 3C). Label the Output Population as “Mitochondria Selected”.

    13. Calculate Intensity Properties (2). Set Channel “Cy3”, Population “Image Region (GFP-neurites)” and Region “Image Region”. Set Method to “Standard” and expand the drop down menu to tick “Mean”. This will measure the TMRM signal intensity within the GFP-neurite selection.

    14. Calculate Morphology Properties. Set Population “Mitochondria Selected” and Method “Standard”. Expand the drop down menu and tick “Area” and “Roundness”. This will measure the morphological properties of the segmented mitochondria. Label Output Properties as “Mitochondria Morphology”.

    15. Calculate Texture Properties. Set Channel “Cy3”, Population “Mitochondria” and use Method “SER Features”. This will measure the texture properties of the segmented mitochondria. Label Output Properties as “Mitochondria Texture”.

    16. Find Nuclei (2). Set Channel “DAPI” and use Method “C”. Expand the drop down menu and adjust the Common Threshold appropriately. Adjust the minimum area to remove background debris (we used > 20 μm2) (Figure 3D). Label the Output Population “Nuclei (DAPI)”.

    17. Define Results. Use Method “List of Outputs”. For each Population, expand the drop down menu and select the type of output you wish from each measured parameter. Alternatively, use “Apply to All” to select an output type for all measured parameters in that population. Click the green arrow at the top of the building block to apply changes and then save the protocol.



    Figure 3. Analysis protocol selections using the Columbus Image Data Storage and Analysis System. A. “Find Neurites” selection using the Alexa 488 channel, see step 4c of the Data analysis protocol. B. “Find Image Region” selection using the Alexa 488 channel, see step 4h. C. “Select Population” using the segmented Mitochondria channel, see step 4l. The indented image shows a close up of the individual selections. D. “Find Nuclei” selection using the DAPI channel, see step 4q. All scale bars = 20 μm.


  5. Select the Batch Analysis tab and select the data folder from the left-hand panel that you wish to analyse. Then, select the analysis protocol written above. Click the green arrow to start the analysis.

  6. When the analysis finishes, the results will be shown in the corresponding data folder in the left-hand panel. Select the Browse tab. Open the corresponding data folder by clicking the plus symbol next to it, and select the “Result” file to open it. In the bottom-right-hand panel, the “Summary” tab will show the data from that analysis. Click the adjacent tab corresponding to the name of the “Result” file to see the raw data in full. Download the file using the blue “Download” link.

  7. Open the downloaded file in Excel 2016. Organise the data so that parameter values for each compound at each concentration as well as control wells are ordered sequentially.

  8. Input the data for each compound into GraphPad Prism 8.2 using a separate data table for each parameter. Ensure each compound is organised by column and each concentration by row. Include the data for positive control compounds.


Statistical analysis

The statistical analysis will depend on the experimental setup. However, some important plate effects should be controlled to ensure robustness and reproducibility of the assay.

  1. Create another data table and plot the nuclei number (DAPI) across the whole plate. This should remain consistent to minimise plate drift effects.

  2. Create another data table and plot the values for DMSO vehicle control wells against DMSO rotenone control wells for each parameter (Figure 4A). A significant difference should be seen for each parameter.

  3. Calculate Z’ and SW scores in order to ensure the robustness of the assay. The formulae are:

    In this protocol, ‘max’ would refer to values from DMSO vehicle control wells, and ‘min’ would refer to values from DMSO rotenone control wells. Example data for Z’ scores for various parameters in this assay are given in Figure 4B. In general, a Z’ score of above 0.5 and an SW score of above 2 are acceptable for use in phenotypic screening assays.

  4. Typically, One-Way ANOVA with Dunnet’s post-hoc test would be performed to compare single concentration drug effects against vehicle treated wells. Alternatively, if a concentration response plate map is undertaken, a Nonlinear regression (curve fit) analysis on the data, using log(agonist) vs. response (three parameters) is typically used. All analysis is carried out in GraphPad Prism 8.2.



    Figure 4. Example data showing rotenone effect, Z’-scores from GFP-LUHMES 384-well plate drug screening. A. Rotenone effect for three example parameters is shown by comparing values for DMSO vehicle-treated wells against DMSO rotenone-treated wells. In both cases, the mean parameter value for rotenone-treated wells is significantly different to vehicle-treated wells (*** = P < 0.0001). B. Z’-scores for several parameters across four separate plates, each performed three weeks apart. The parameter ‘Neurite Roundness’ represents a measure of cell shape by comparing cell area to cell perimeter; a healthy neuron with extensive neurites would have a very low score approaching 0, compared to a spherical cell which should score up to 1.

Notes

  1. GFP-LUHMES cells should be retired once they reach passage 15 and replaced with younger stocks.

  2. When freezing GFP-LUHMES stocks, we recommend to split a fully confluent T75 flask, resuspend the pellet in 5 ml LUHMES Proliferation Media, add 10% DMSO and 20% FBS, then divide equally between 5 cryovials.

  3. When counting GFP-LUHMES cells on the haemocytometer, only circular, bright spheres should be counted.

  4. When transferring the differentiated GFP-LUHMES cells into the 384-well plate, act quickly and re-homogenise the cell suspension in the reservoir using the multichannel pipette between transfers. This is to ensure the number of cells plated per well is as consistent as possible across the plate.

  5. The analysis software can output several additional parameters, including cell number and number of cells with neurites which can be useful parameters to assess cell viability, drift across plates and conditions as well as variability between assay days.

Recipes

  1. Sterile 1x PBS

    1. Add 5 PBS tablets to 500 ml of distilled water in a flask suitable for autoclaving

    2. Mix until tablets dissolve and autoclave

    3. Store at room temperature

  2. Sterile 1x Trypsin

    1. Add 500 ml of distilled water to a flask suitable for autoclaving

    2. Autoclave the flask and transfer to a sterile culture hood

    3. In the hood, add 45 ml of sterile distilled water to 5 ml of Trypsin 10x in a 50 ml Falcon

    4. Mix by inverting 2-3 times

    5. Store at 4 °C

  3. LUHMES Base Media

    Store at 4 °C

    500 ml Advanced DMEM/F-12

    5 ml N-2 Supplement

    5 ml Pen-Strep

    5 ml L-Glutamine

  4. LUHMES Proliferation Media

    Store for one week at 4 °C

    50 ml LUHMES Base Media

    40 ng/ml FGF-basic

  5. LUHMES Differentiation Media

    Prepare fresh for each use

    20 ml LUHMES Base Media

    1 μg/ml Tetracycline

    1 mM dCAMP

    2 ng/ml GDNF

Acknowledgments

This work was funded by a Parkinson’s UK Senior Fellowship award to HM (F-1301) and a Parkinson’s UK Virtual Biotech project grant to HM (I-1904). LF is supported by the Academy of Medical Sciences (SBF002\1142) and the Medical Research Council (MRC, grant 1812144). IV was supported by Consejo Nacional de Ciencia y Technologia (CONACyT), Mexico.

The generation of GFP-LUHMES cells was based on a previously established protocol from Ratcliffe et al., 2018.

Competing interests

The authors declare no competing interests.

References

  1. Armstrong, M. J. and Okun, M. S. (2020). Diagnosis and Treatment of Parkinson Disease: A Review. JAMA 323(6): 548-560.
  2. Delp, J., Funke, M., Rudolf, F., Cediel, A., Bennekou, S. H., van der Stel, W., Carta, G., Jennings, P., Toma, C., Gardner, I., van de Water, B., Forsby, A. and Leist, M. (2019). Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants. Arch Toxicol 93(6): 1585-1608.
  3. Dolga, A. M., de Andrade, A., Meissner, L., Knaus, H. G., Höllerhage, M., Christophersen, P., Zischka, H., Plesnila, N., Hoglinger, G. U. and Culmsee, C. (2014). Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons. Cell Death Dis 5: e999.
  4. Hauser, D. N. and Hastings, T. G. (2013). Mitochondrial dysfunction and oxidative stress in Parkinson's disease and monogenic parkinsonism. Neurobiol Dis 51: 35-42.
  5. Höllerhage, M., Moebius, C., Melms, J., Chiu, W.-H., Goebel, J. N., Chakroun, T., Koeglsperger, T., Oertel, W. H., Rösler, T. W., Bickle, M. and Höglinger, G. U. (2017). Protective efficacy of phosphodiesterase-1 inhibition against alpha-synuclein toxicity revealed by compound screening in LUHMES cells. Sci Rep 7(1): 11469.
  6. Krug, A. K., Balmer, N. V., Matt, F., Schonenberger, F., Merhof, D. and Leist, M. (2013). Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants. Arch Toxicol 87(12): 2215-2231.
  7. Lotharius, J., Barg, S., Wiekop, P., Lundberg, C., Raymon, H. K. and Brundin, P. (2002). Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J Biol Chem 277(41): 38884-38894.
  8. Lotharius, J., Falsig, J., van Beek, J., Payne, S., Dringen, R., Brundin, P. and Leist, M. (2005). Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci 25(27): 6329-6342.
  9. Park, J. S., Davis, R. L. and Sue, C. M. (2018). Mitochondrial dysfunction in parkinson's disease: new mechanistic insights and therapeutic perspectives. Curr Neurol Neurosci Rep 18(5): 21.
  10. Mortiboys, H., Aasly, J. and Bandmann, O. (2013). Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson's disease. Brain (10): 3038-50.
  11. Yamaguchi, A., Ishikawa, K. I., Inoshita, T., Shiba-Fukushima, K., Saiki, S., Hatano, T., Mori, A., Oji, Y., Okuzumi, A., Li, Y., Funayama, M., Imai, Y., Hattori, N. and Akamatsu, W. (2020). Identifying Therapeutic Agents for Amelioration of Mitochondrial Clearance Disorder in Neurons of Familial Parkinson Disease. Stem Cell Reports 14(6): 1060-1075.
  12. Poewe, W., Seppi, K., Tanner, C. M., Halliday, G. M., Brundin, P., Volkmann, J., Schrag, A. E. and Lang, A. E. (2017). Parkinson disease. Nat Rev Dis Primers 3: 17013.
  13. Ratcliffe, L. E., Vázquez Villaseñor, I., Jennings, L., Heath, P. R., Mortiboys, H., Schwartzentruber, A., Karyka, E., Simpson, J. E., Ince, P. G., Garwood, C. J. and Wharton, S. B. (2018). Loss of IGF1R in human astrocytes alters complex I activity and support for neurons. Neuroscience 390: 46-59.
  14. Scholz, D., Pöltl, D., Genewsky, A., Weng, M., Waldmann, T., Schildknecht, S. and Leist, M. (2011). Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem 119(5): 957-971.
  15. Stiegler, N. V., Krug, A. K., Matt, F. and Leist, M. (2011). Assessment of chemical-induced impairment of human neurite outgrowth by multiparametric live cell imaging in high-density cultures. Toxicol Sci 121(1): 73-87.
  16. Tong, Z. B., Hogberg, H., Kuo, D., Sakamuru, S., Xia, M., Smirnova, L., Hartung, T. and Gerhold, D. (2017). Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening. J Appl Toxicol 37(2): 167-180.

简介

[摘要]帕金森氏病是一种破坏性神经退行性疾病,影响65岁以上人口的2-3%。目前尚无改善疾病的治疗方法。帕金森氏病的主要病理特征之一是线粒体功能障碍,许多工作旨在鉴定可恢复破坏的线粒体生理的治疗性化合物。然而,在疾病相关模型中对线粒体功能障碍进行建模,适用于筛选大型化合物文库的改善作用,这是一个巨大的挑战。病人原代细胞,SHSY-5Y细胞和体内 帕金森氏病模型被广泛用于研究线粒体功能障碍在帕金森氏症中的作用。确实,许多研究已经利用LUHMES细胞研究帕金森氏病,但是,尽管与其他常用模型相比,LUHMES细胞与其他常用模型相比具有多种优势,例如快速分化和高均一性,但以前并未用作PD相关的线粒体功能障碍的复合筛选模型。 (例如,与来自iPSC的神经元相反),以及与人类中脑组织相关的生理学,能够分化为高度表达特征性标记的多巴胺能样神经元。在先前产生GFP + -LUHMES细胞以模拟代谢功能障碍后,我们报道了在PD相关的线粒体功能障碍恢复模型中使用GFP + -LUHMES细胞进行高通量化合物筛选的方案。该协议描述了通过评估一系列线粒体和神经元形态学参数,使用强大且可重现的毒素诱导的GFP + -LUHMES细胞模型进行高通量化合物筛选的方法。我们还提供了有关数据和统计分析的详细说明,包括Z'得分的示例计算,以评估独立实验中的统计效应大小。


[背景]帕金森氏病(PD)是一种神经退行性疾病,其主要特征是中脑黑质中多巴胺能神经元的丢失以及神经元内包涵体中α-突触核蛋白的积累。它是第二种最常见的神经退行性疾病,影响65岁以上人口中2-3%的人口(Poewe et al。,2017)。患者表现为静息性震颤,运动迟缓和肌肉僵硬,以及非运动性症状,包括抑郁,失眠和记忆障碍。当前,尚无可预防或延缓PD进展的疾病缓解疗法,这迫切需要有效的治疗方法(Armstrong和Okun,2020年)。

线粒体功能障碍是PD的关键病理标志。与家族性PD相关的许多遗传基因座都编码参与线粒体功能或调控的蛋白质(Hauser and Hastings,2013)。PD病理学中多种线粒体途径受到干扰,包括ATP产生,线粒体吞噬,运输,生物发生和钙缓冲作用(Park等人,2018)。因此,毫不奇怪的是,线粒体功能障碍是PD中治疗发现的流行靶标,其目的是鉴定能够增强那些在疾病状态下受干扰的线粒体途径,使其恢复正常生理水平的化合物。这提出了寻找一种有效的体外模拟线粒体功能障碍的方法的挑战,该方法可以在疾病相关模型中有效地筛选大型化合物库。

我们先前曾报道使用原发性患者成纤维细胞来筛选可挽救那些成纤维细胞中见到的线粒体表型的化合物(Mortiboys等人,2013)。此外,最近描述了使用高通量,半自动成像系统的患者来源的iPSC神经元模型,以鉴定可减轻线粒体清除缺陷的化合物(Yamaguchi等人,2020年)。这两种系统都利用患者来源的细胞,使用患者来源的模型时一个潜在的问题是所需的材料量和样品的均匀性。替代方案是使用隆德人中脑(LUHMES)细胞。LUHMES细胞是MESC2.10细胞系的亚克隆,衍生自人胚胎腹侧中脑组织,并通过整合可被四环素调节的v- myc逆转录病毒因子而永生(Lotharius等人,2002和2005)。简而言之,LUHMES细胞以增殖状态存在,直到加入四环素,二丁酰环AMP(dCAMP )和神经胶质细胞衍生的神经营养因子(GDNF)阻止增殖并诱导均匀分化为多巴胺能样表型。由于PD主要影响黑质的多巴胺能神经元,因此分化的LUHMES细胞表型在模型上与生理高度相关。进一步的表征表明,分化的LUHMES细胞在许多方面类似于原代神经元培养:神经元标志物的广泛上调,广泛的神经突向外生长和基本的电生理特征(Scholz等,2011)。与许多其他转化的神经元细胞系不同,处于分化状态的LUHMES细胞已关闭c- myc ,并且未见细胞周期失调。当考虑涉及神经元DNA修复和神经变性建模的细胞周期机制时,这一点尤其重要。与原代神经元通常反复无常和表型可变的培养相比,LUHMES细胞易于处理,均匀且在分化诱导后纯度高(> 99%),因此可用于高通量筛选(Scholz等人,2011)。一项比较研究还显示,与其他两种常用的神经元模型SH-SY5Y神经母细胞瘤细胞和人胎儿神经干细胞相比,LUHMES细胞表达更高水平的神经元标记(TUBB3,ENO2,MAP2 ),并且具有更高的神经毒性敏感性(Tong等。,2017)。

LUHMES细胞已被广泛用于高通量筛选中,以鉴定神经毒剂(Stiegler等,2011; Krug等人,2013),包括一种针对线粒体毒性的特异药物(Delp等,2019)。但是,迄今为止,我们仅发现一项研究,该研究在PD治疗发现中利用了LUHMES细胞的高通量潜力。Höllerhage等。使用先前开发的α-突触核蛋白LUHMES模型,通过测量治疗后的细胞活力来筛选1,600种FDA批准的药物具有保护作用(Höllerhage等,2017)。以前,我们报道了GFP + -LUHMES的产生以及它们在与星形胶质细胞共培养中建模代谢功能障碍时的用途(Ratcliffe et al。,2018)。

在这里,我们利用先前生成并验证为代谢功能异常的有用模型的GFP + -LUHMES ,提出了一种高通量筛选化合物的详细且可重现的方案,用于在与PD相关的毒素诱导的分化LUHMES细胞模型中进行化合物的高通量筛选。通过使用高含量的活细胞成像和分析来评估线粒体参数和神经元形态,该协议对以前的研究进行了改进。

在此所述的方法中,根据先前的研究(Krug等人,2013 ;Dolga等人,2014), 用鱼藤酮处理分化的LUHMES细胞以模拟PD相关的线粒体功能障碍和神经元丢失。LUHMES细胞在添加分化因子之前生长至完全融合,然后在两天后重新铺入384孔板。在实验的整个过程中,细胞保留在分化培养基中。在第五天用鱼藤酮处理该板,然后在第六天用测试化合物处理该板,并在第七天进行实时成像(图1)。设计用于筛选的毒素诱导模型时;通常有两种范例。第一种是保护模型,其中潜在的有益化合物在毒素之前添加到细胞中。其次,在该协议中就是这种情况,我是一个恢复模型。先添加毒素,然后再添加潜在的有益化合物。这是测试有益的化合物以恢复已经存在的缺陷,而不是保护其免受损坏。该技术的优势在于,相对容易和快速地生成了可用于大规模化合物筛选的与疾病相关的人多巴胺能神经元的统一种群。此外,高含量的活细胞成像和分析可以评估化合物对广泛的线粒体和神经元形态学参数的剂量反应效应。


图1. GFP-LUHMES药物筛选方案的时间表

关键字:帕金森氏病, 线粒体, 化合物筛选, LUHMES


材料和试剂
用料
384孔黑色板(Greiner-Bio,目录号:781091)
384孔LDV源板(Labcyte ,目录号:LP-0200)
384孔源板密封件(Fluidx ,目录号:41-1011)
5 ml血清移液管(Fisher Scientific,目录号:13-676-10H)
10 ml血清移液管(Fisher Scientific,目录号:13-676-10J)
25 ml血清移液管(Fisher Scientific,目录号:13-678-11)
10个微升移液管尖端(Fisher Scientific公司,目录号:02-707-441)
200个微升移液管尖端(Fisher Scientific公司,目录号:02-707-422)
1 ,000微升移液管尖端(Fisher Scientific公司,目录号:02-707-402)
15 ml F alcon管(Greiner-Bio,目录号:188271)
50 ml F alcon管(Greiner-Bio,目录号:227261)
T75烧瓶(Greiner-Bio,目录号:658175)
冷冻小瓶(Greiner-Bio,目录号:122261)

试剂种类


LUHMES细胞(ATCC ® CRL-2927 TM )
GFP-LUHMES细胞(RRID:CVCL_B056,Ratcliffe等,2018)
表达GFP的慢病毒颗粒
先进的DMEM / F-12(Thermo Scientific,目录号:12634010)
纤连蛋白(Sigma-Aldrich,目录号:FC010)
聚L-鸟氨酸(Sigma-Aldrich,目录号:P4957)
PBS片剂(Thermo Scientific,目录号:BR0014G)
胰蛋白酶10x(Lonza,货号:BE02-007E)
L-谷氨酰胺(Lonza,货号:BE-17-605E)
N-2补充剂(Gibco,目录号:11520536)
Pen-Strep(Lonza,货号:DE17-603E)
碱性FGF(Peprotec ,目录号:100-18B)
dCAMP (Sigma-Aldrich,目录号:D0627)
GDNF(Peprotech ,目录号:450-10)
四环素(Sigma-Aldrich,目录号:T7660)
Hoechst(Sigma,目录号:94403)
TMRM(Invitrogen,货号:T668)
无菌1x PBS(请参阅食谱)
无菌1x胰蛋白酶(请参阅食谱)
LUHMES基本媒体(请参阅食谱)
先进的DMEM / F-12                         
N-2补品                         
Pen-Strep                                       
谷氨酰胺


LUHMES扩散介质(请参阅食谱)
LUHMES基础媒体                         
碱性FGF


LUHMES差异化媒体


四环素


营地                                                     
GDNF



设备



机械移液枪,用于血清移液器
机械移液器(P20,P200,P1000)
多通道机械移液器(P10,P200)
4°C冰箱
-20°C冷冻室
-80°C冷冻室
无菌组织培养罩
高压釜
组织培养离心机(MSE Harrier 15/80,目录号:MSB080.CX1.5)
源板离心机
CO 2培养箱(三洋,型号:MCO-19AIC)
Echo 550液体处理器(Labcyte ,目录号:Echo 550)
MultiPod控制器(Roylan Developments,目录号:SPOD0012)
StoragePod机柜(Roylan Developments,目录号:SPOD0010)
Opera Phenix高内涵放映系统(PerkinElmer)

软件



回声液体处理器软件(Labcyte )
回声板重新格式化(Labcyte )
和谐高内涵成像和分析软件(PerkinElmer)
哥伦布图像数据存储和分析系统(PerkinElmer)
Excel 2016(Microsoft)
GraphPad Prism 8.2(GraphPad)


程序



GFP-LUHMES细胞的生成


GFP-LUHMES细胞是通过在PGK启动子的控制下用表达GFP的慢病毒颗粒转导增殖LUHMES细胞而产生的(Ratcliffe等,2018)。


每个T75烧瓶以1.5 x 10 6的密度播种LUHMES细胞(p3-p5)。
用表达GFP的慢病毒颗粒转导24小时。MOI为8;为了进行转导,使用1:100的稀释液。先前通过FACS分选评估转导效率为98.5%。表达GFP的慢病毒颗粒是谢菲尔德大学的Eva Karyka和Mimoun Azzouz的礼物。
24小时后,在开始实验前,在LUHMES增殖培养基中保持72小时。

第一部分:GFP-LUHMES细胞维持方案



在无菌培养罩中去除和接种GFP-LUH MES细胞
根据以下食谱准备LUHMES增殖培养基和无菌1x PBS。
准备一个纤连蛋白聚-L-鸟氨酸涂层的T75烧瓶。
通过加入制备出10毫升纤连蛋白聚L-鸟氨酸涂布每T75瓶解的1微克/毫升纤连蛋白和50微克/ ml的聚-L-鸟氨酸到无菌蒸馏水中。  
混合纤连蛋白聚-L-鸟氨酸涂层溶液,并使用10毫升血清移液器将10毫升转移至T75烧瓶中。
将烧瓶与纤连蛋白聚-L-鸟氨酸涂层溶液在37°C下孵育至少3小时或过夜。
使用10毫升血清移液管从烧瓶中除去纤连蛋白聚L-鸟氨酸涂层溶液。
使用10 ml血清移液器将5 ml无菌1x PBS转移到烧瓶中,并从一侧到另一侧轻轻倾斜烧瓶,使PBS遍及整个表面,从而清洗涂层表面。应当将PBS留在烧瓶中,直到将细胞种入烧瓶之前,以防止涂层干燥。烧瓶在使用前最多可以准备1小时。
将GFP-LUHMES细胞解冻并接种到LUHMES增殖培养基中。
从冰箱中取出准备好的LUHMES增殖介质,然后加热至室温。
从冷藏库中取出一个冷冻的GFP-LUHMES细胞小瓶。通常,我们建议重振1-10代的小瓶,以使存活的细胞可以经历多次分裂,然后在15代退休。
使用10毫升血清移液器将10毫升室温LUHMES增殖培养基添加到15毫升F alcon中。
转移〜500 μ升室温LUHMES扩散介质向GFP-LUHMES细胞小瓶使用P1000机械移液管慢慢通过抽吸和反复排出液体在尖端混合。
将GFP-LUHMES细胞小瓶中的溶液完全除霜后,将其转移至15 ml F alcon中的室温LUHMES增殖培养基中。
从带涂层的T75烧瓶中取出所有1x PBS,然后使用10 ml血清移液管将15 ml F alcon中的所有细胞溶液添加到T75烧瓶中。
前后倾斜烧瓶,然后左右倾斜2-3次,以使GFP-LUHMES细胞均匀分布在涂层表面上。
将烧瓶在37°C / 5%CO 2培养箱中孵育。
在第二天,更换GFP-LUHMES细胞培养基以去除所有未完全附着的细胞。
使用10毫升血清移液器从T75烧瓶中取出所有培养基。
使用10毫升血清移液器将10毫升室温LUHMES增殖培养基添加到T75烧瓶中。
将烧瓶在37°C / 5%CO 2培养箱中孵育。

每3-4天或在无菌培养柜中汇合率为80-90%时分裂GFP-LUHMES细胞
根据上述步骤(步骤A2),准备两个纤连蛋白聚L-鸟氨酸涂层T75烧瓶。
镨根据下面的食谱epare LUHMES扩散媒体和1x胰蛋白酶。如果已预先准备好,请从冰箱中取出,使其温度升至室温。
将GFP-LUHMES细胞分成两个装有LUHMES增殖培养基的烧瓶。一瓶用于维持种群,一瓶用于区分。
使用10 ml血清移液管从GFP-LUHMES细胞瓶中取出所有LUHMES增殖培养基,并将5 ml转移到15 ml F alcon中。
使用10 ml血清移液器将5 ml室温1x胰蛋白酶添加到GFP-LUHMES细胞烧瓶中。
在37°C / 5%CO 2培养箱中孵育GFP-LUHMES细胞烧瓶5分钟。
用力敲打GFP-LUHMES细胞瓶的侧面2-3次,以将细胞移入溶液中。如果成功,细胞云应该在解决方案中可见。
将所有细胞溶液从GFP-LUHMES细胞瓶转移到含有5 ml用过的LUHMES增殖培养基的15 ml F alcon中。
在工作台上,将15 ml F alcon以400 xg离心4分钟。
使用10毫升血清移液器移出上清液并丢弃。
根据所需的分裂比例,将沉淀的GFP-LUHMES细胞重悬于适量的LUHMES增殖培养基中。我们建议完全融合的烧瓶的分流比为1:15,准备在3天后再次分流,尽管这可能需要根据细胞行为而稍作调整。
从包被的T75烧瓶中取出所有1x PBS,并使用10 ml血清移液管向每个烧瓶中加入10 ml LUHMES增殖培养基。
向两个T75烧瓶中的LUHMES增殖培养基中添加适量的重悬的GFP-LUHMES细胞溶液。

每两天更换一次GFP-LUHMES细胞上的增殖培养基(拆分后)
删除˚F爱尔康从冰箱里LUHMES扩散媒体的管温至室温。
将GFP-LUHMES细胞上的培养基更改为新鲜的增殖培养基。
使用10 ml血清移液管从GFP-LUHMES细胞瓶中取出所有LUHMES增殖培养基,并丢弃。
使用10毫升血清移液器将10毫升室温LUHMES增殖培养基添加到GFP-LUHMES细胞烧瓶中。
将烧瓶在37°C / 5%CO 2培养箱中孵育。

第二部分:GFP-LUHMES细胞分化和重铺方案



开始分化GFP-LUHMES细胞(第1天)
GFP-LUHMES细胞分裂后等待2-3天,以使其增殖。
根据以下配方准备LUHMES分化培养基。每次使用前必须将其新鲜。
将GFP-LUHMES细胞上的增殖培养基更改为分化培养基。
使用10 ml血清移液管从GFP-LUHMES细胞瓶中取出所有LUHMES增殖培养基,并丢弃。
使用10毫升血清移液器将10毫升室温LUHMES分化培养基添加到GFP-LUHMES细胞烧瓶中。
将烧瓶在37°C / 5%CO 2培养箱中孵育。

Replate区分GFP-LUHMES到384孔PLA TE (目录号:781091) (第3天)
准备纤连蛋白聚-L-鸟氨酸包被的384孔板。
制备15毫升纤连蛋白聚L-鸟氨酸涂布每384解-通过加入1孔板微克/毫升纤连蛋白和50微克/ ml的聚-L-鸟氨酸到无菌蒸馏水中。
混合纤连蛋白聚-L-鸟氨酸涂层溶液,并将15毫升转移到适合多通道移液的合适容器中(例如,但不限于上翘的移液箱盖,干净且无菌)。
使用P200多通道机械移液器将50μl纤连蛋白聚L-鸟氨酸涂层溶液转移至384孔板的每个孔中,除了最外面的两个列和行。
使用P200多通道机械移液器将40μl无菌1x PBS转移至每个空的最外面的孔中,以充当防火墙。
将烧瓶与纤连蛋白聚-L-鸟氨酸涂层溶液在37°C下孵育至少3小时或过夜。
使用P200多通道机械移液器从384孔板中除去纤连蛋白聚L-鸟氨酸涂层溶液。
使用P200多通道机械移液器将50μl无菌PBS转移至每个包被孔中,以清洗包被表面。
根据以下配方准备LUHMES分化培养基。
提起GFP-LUHMES细胞并将10,000个细胞/孔接种到包被的384孔板中。
使用10 ml血清移液管从分化的GFP-LUHMES细胞瓶中取出所有LUHMES分化培养基,并将5 ml转移到15 ml F alcon中。
使用10 ml血清移液管将5 ml室温1x胰蛋白酶添加到分化的GFP-LUHMES细胞烧瓶中。
在37°C / 5%CO 2培养箱中孵育分化的GFP-LUHMES细胞烧瓶5分钟。
用力敲打分化的GFP-LUHMES细胞瓶的侧面2-3次以将细胞移入溶液中。
将所有细胞溶液从分化的GFP-LUHMES细胞烧瓶转移到含有5 ml用过的LUHMES分化培养基的15 ml F alcon中。
在工作台上,将15 ml F alcon以400 xg离心4分钟。
使用10毫升血清移液器移出上清液并丢弃。
将沉淀的GFP-LUHMES细胞重悬于5 ml的LUHMES分化培养基中,通过抽吸并用P1000机械移液器将GFP-LUHMES重悬10-15次,使其充分匀浆。
制备血球和转移10微升GFP-LUHMES再悬浮到计数网格。
计数GFP-LUHMES细胞的数量,并计算GFP-LUHMES重悬液中的细胞/ ml。
除去含有4 x 10 6个细胞的GFP-LUHMES重悬液,并转移至15 ml F alcon。这将是电镀悬浮液。
使用10毫升血清移液管,用LUHMES分化培养基将平板悬浮液加至最终体积12毫升。
使用P200多通道机械移液器从384孔板前半部分的包被孔中除去PBS。
通过用10 ml血清移液管吸移溶液10-15次,将镀液悬浮液彻底匀浆。
将一半的电镀悬浮液(6毫升)转移到适合多通道移液的合适容器中。
将30μl的平板悬浮液转移至384孔板的每个包被孔中。
使用P200多通道机械移液器从384孔板后半部分的包被孔中除去PBS。
通过用10 ml血清移液管吸出并排出溶液10-15次,将镀液悬浮液在15 ml F alcon中彻底重新均匀化。
将其余的电镀悬浮液转移到储槽中,并使用P200多通道机械移液器将每孔30μl的电镀悬浮液填充到剩余的涂覆孔中。
来回移动板数次,然后左右移动数次,以确保细胞均匀分布在孔底。
在37°C / 5%CO 2培养箱中孵育384孔板。

第三部分:GFP-LUHMES药物筛选方案(第二部分的时间表继续)



用鱼藤酮处理分化的GFP-LUHMES 384孔板(第5天)
根据以下配方准备LUHMES分化培养基。
在无菌培养罩中准备鱼藤酮处理过的培养基和媒介处理过的培养基。
为了避免细胞的干扰,我们避免移除媒体,而是补足每孔10微升鱼藤酮处理的介质给予40个的最终体积微升媒体每孔。因此,鱼藤酮处理过的培养基必须以所需最终浓度(4μM )的四倍制备。  
将4 ml LUHMES分化培养基转移至15 ml F alcon并贴上“鱼藤酮”标签。转移4毫升LUHMES分化培养基的另一15毫升˚F爱尔康和标签“车辆”。
准备在乙醇中的10 mM鱼藤酮。鱼藤酮粉应在通风橱中称量。
添加6.4微升10毫鱼藤酮向媒体在15毫升˚F爱尔康标记“鱼藤酮”使用P20移液管机械。孔中鱼藤酮的最终浓度为4μM 。
使用P20机械移液器将等体积的乙醇(6.4μl )加入15 ml F标记为“ Vehicle”的F alcon 。
缓慢颠倒3-4次,混合15 ml F alcons。
将处理过的培养基转移到分化的GFP-L UHMES 384孔板中。我们建议的板地图如图URE 2A(即,“DMSO媒介物”孔用载体处理的媒体,而所有其他井用鱼藤酮处理的培养基处理处理过的)。
转移的“鱼藤酮”媒体内容˚F爱尔康和“车辆”媒体˚F爱尔康成多通道移液单独的存储器。相应地标记容器。
使用P10多通道机械移液器将10μl鱼藤酮处理过的培养基转移至384孔板的每个孔中,确保移液器吸头放置在孔中足够深,以确保喷射的液体与孔中的内容物混合。保留一些未处理的孔,以添加溶媒处理的介质,以稍后测量鱼藤酮的效果。我们建议按照以下步骤B3的说明,在每个仅DMSO的控制栏中包括对某些井进行车辆处理。使用P10多通道机械移液器将10μl媒介物处理的介质转移到384孔板的每个未处理孔中。
在37°C / 5%CO 2培养箱中孵育384孔板。
在DMSO中准备化合物并编写协议以使用Echo 550 Liquid Handler和Opera Phenix高内涵筛选系统
在带有板密封的384孔离子源板上(目录号:LP-0200)在100%无水DMSO中准备化合物和文库。将其存放在StoragePod机柜中,并使用MultiPod控制器保持干燥的氮气氛围。
使用Echo Liquid Handler软件编写复合传输协议。源板应设置为“ 384LDV-DMSO”,目标板应设置为“ Griener_384PS_781096”,并选择自定义映射。
设置方案,以将适当体积的样品从源板的每个孔转移到目标板上的相应孔,以提供所需的最终药物浓度。我们建议将目标板分成四等分,并使用每四分之一的第一列进行仅DMSO处理作为阴性对照(图2)。然后可以将每个季度中每个经过化合物处理的井的参数值标准化为该季度中各个DMSO对照井的值。对于一个完整的剂量反应曲线,我们建议测试8个浓度对于每种化合物在每个半对数,即,3纳米,10纳米,30纳米,100纳米,300纳米,1 ,000 nM的,3 ,000纳米,10 ,000 nM 。我们还建议在每个平板上至少包含一种阳性对照化合物。我们使用了先前在室内发现的对线粒体和形态参数显示出有益影响的化合物。



图2.测定GFP-LUHMES 384孔板药物筛选后的板图和示例图像。A.板图显示了对照孔和测试化合物孔的建议布置。除“ DMSO载剂”井外,所有井均用鱼藤酮处理。的颜色表示在浓度nM的每种化合物。BD。蓝色= DAPI,橙色= TMRM,绿色= GFP,比例尺s = 20μm 。使用Opera Phenix高内涵筛选系统从(B)车辆处理的DMSO对照井,(C)鱼藤酮处理的DMSO对照井和(D)鱼藤酮处理的阳性对照井( 1 ,000纳米)。



在Harmony High-Content Imaging and Analysis Software上编写用于对LUHMES细胞进行成像的成像方案(详细信息如下)。使用DAPI,Alexa 488和Cy3通道分别捕获Hoechst信号,GFP信号和TMRM信号。使用测试图像以确保每个激光的适当曝光时间;这需要由用户设置,因为它取决于每个成像系统的激光功率的信号强度。使用40x水共焦物镜,将场数设置为10,将堆栈数设置为6。每个孔中的字段。

用化合物处理分化的GFP-LUHMES 384孔板(第6天)
卸压的StoragePod使用机柜多脚外壳并取出384孔源板。
以2500 xg的速度离心384孔源板2分钟,以除去板内容物中的所有气泡。
在每次独立印版拍摄之前,请校准并专注于Echo 550液体手柄r。
使用Echo 550液体处理机测量384孔ldv源板。确保每个孔中的体积足够高,以根据化合物转移规程将所需体积转移至目标板。请注意,384孔源头中每个孔的体积不应超过12μl 。确保每个孔中的水含量不超过30%(这是根据DMSO含量的测量得出的,水含量不应低于70%;如果是这种情况,则必须刷新该孔中的水含量) 。
从384孔离子源板上取下密封垫,然后启动化合物转移方案。出现提示时,将384孔源板和目的板转移到机器中。
方案完成后,取出目的板并立即在37°C / 5%CO 2的培养箱中培养。
更换384孔离子源板上的密封,并使用Multipod Controller对StoragePod机柜减压。装回StoragePod机柜中的384孔源板。锁定和repressurise的StoragePod使用机箱多脚控制器。

测定并成像GFP-LUHMES 384孔板(第7天)
根据以下配方准备LUHMES分化培养基。
准备测定培养基。
在无菌蒸馏水中准备10 mM Hoechst。
准备80 μM TMRM在DMSO。
将4 ml LUHMES分化培养基转移到15 ml F alcon中。
20添加微升的X毫Hoechst的解决15毫升˚F爱尔康。
20添加微升的X毫TMRM解决15毫升˚F爱尔康。
通过缓慢颠倒F alcon 4-5次来混合15 ml F alcon(测定培养基)的内容物。
用检测培养基孵育GFP-LUHMES 384孔板1小时。
将测定介质转移到适合多通道移液的合适容器中。
使用P10多通道机械移液器将10μl分析介质转移到384孔板的每个孔中,确保移液器吸头放置在孔中足够深,以确保喷射的液体与孔中的内容物混合。
将GFP-LUHMES 384孔板在37°C / 5%CO 2培养箱中孵育1小时。
使用Opera Phenix高内涵筛选系统对GFP-LUHMES 384孔板成像。
将Opera Phenix上的TC O(温度和CO 2 )设置设置为37°C和5%CO 2并将两者都设置为“ On”。出现提示时,打开连接的CO 2气瓶。使系统达到所需的温度和气体水平。
打开准备的成像协议。
加载GFP-LUHMES 384孔板。
冲洗水物镜。
运行测试并调整每个通道的曝光时间,以使强度在背景水平上显着。如果需要,请调整堆栈的高度,以使图像在单元的整个高度上都能很好地聚焦。
运行成像协议(图2)。

数据一nalysis



                            图像将使用哥伦布图像数据存储和分析系统进行批量分析。GFP信号将用于评估神经元形态参数,TMRM信号将用于评估线粒体功能和形态。


提供了一个简短的视频(视频1),以显示分析软件并提供协议部分的指南,并在下面提供详细说明。



视频1.使用哥伦布分析软件进行分析设置



成像协议完成后,将测量文件导出到方便的位置。确保选择了“ Measurements Inc.关联文件”,并记下导出路径。
将导出的文件导入到Columbus图像数据存储和分析系统。
选择“图像分析”选项卡,然后打开导入的文件。
使用以下构造块编写分析协议。通过单击蓝色加号添加新的构建基块。
输入图像。这是将图像加载到分析工具中的位置。选择“最大投影”进行堆叠处理,选择“基本”进行平面校正。取消选中“快速调谐”旁边的框。
找到核子。此第一个“查找核”构建基块用于辅助后续构建基块中的GFP神经突检测。Hoechst信号将在以后用于更准确地计数原子核。将通道设置为“ Alexa 488”(GFP)并使用方法A。将输出种群标记为“核仁(GFP神经突)”。
查找神经突。将通道设置为“ Alexa 488”(GFP),并确保将“人口”设置为“核(GFP神经突)”,将“区域”设置为“核”。将进行两项选择,如图像上方的标签所示。上面选择的核的位置,并标记为“核(GFP神经突)”(图3A),第二个检测图像中标记为“神经核节段”的任何神经突结构。单击黑色的向下箭头并调整值。每个检测参数,以便尽可能多地选择神经突,不要在“神经链片段”选择中关注背景检测,因为在以下构造块中将删除该背景检测,将输出总体标记为“神经链片段”。
选择人口。现在,我们将滤除上面“查找神经突”选择中检测到的背景碎片。将“人口”设置为“ Neurite细分”,将“方法”设置为“按属性过滤”。单击黑色的向下箭头,展开下拉菜单。选择“段长”,“神经节段面积”和“神经节段圆度”,并调整值,直到从选择中去除了大部分碎片,但“真实”神经突保持选中状态。我们使用的值> 12.5,> 0.7 ,分别> 0。将输出总体标记为“选定的神经链段”。
计算形态特性。选择人口“选择了Neurite段”并使用方法“标准”。展开下拉菜单,然后选中每个参数旁边的框。
计算形态学性质(2)。选择种群“ Nuclei(GFP神经突)”,区域“ Neurite树”和方法“标准”。展开下拉菜单,然后选中每个参数旁边的框。
查找图像区域。选择通道“ Cy3”,将“ ROI填充”设置为“无”,将方法设置为“通用阈值”。展开下拉菜单,然后将阈值更改为适当的水平,以选择大部分信号而不会产生过多背景。嘀“拆分成对象”,展开进一步下拉菜单和最小区域改变为适当的值,以除去背景(我们使用> 25微米2 )。保持“填充孔”不变。将输出人口标记为“图像区域(Cy3)”。
查找图像区域(2)。重复步骤(g。),但使用“ Alexa 488”通道选择GFP神经突图像区域(图3B)。将输出种群标记为“图像区域(GFP神经突)”。
过滤图像。现在将分割线粒体。将通道“ Cy3”和方法设置为“ Texture SER”。展开下拉菜单,然后将“过滤器”设置为“ SER Ridge”,将“缩放”设置为1 px ,将“归一化”设置为“未归一化”。将输出图像标记为“ SER Ridge”。
计算图像。该步骤放大信号以改善分割。将方法设置为“按公式”,然后输入公式“ A * 1000”。将通道A设置为“ SER Ridge”,并将输出图像标记为“计算图像”。
查找景点。将“计算图像”通道和“ ROI填充”设置为“图像区域(GFP神经突)”。这样可以确保仅将GFP神经突选择中的线粒体进行分割。将方法设置为“ D”,并将输出人口标记为“线粒体”。
选择人口。将人口设置为“线粒体”,将方法设置为“按属性过滤”。选择参数“ Spot Area [px 2 ]”,选择“>”,然后输入一个最小值以清除选择中的任何碎片(我们使用8 px 2 )(图3C)。将输出人口标记为“选择的线粒体”。
计算强度特性(2)。设置通道“ Cy3”,种群“图像区域(GFP神经突)”和区域“图像区域”。将“方法”设置为“标准”,然后展开下拉菜单以勾选“均值”。这将在GFP神经突选择中测量TMRM信号强度。
计算形态特性。设置人口“选择线粒体”和方法“标准”。展开下拉菜单,然后勾选“区域”和“圆度”。这将测量分段线粒体的形态学特性。将输出属性标记为“线粒体形态”。
计算纹理属性。设置通道“ Cy3”,人口“线粒体”,并使用方法“ SER功能”。这将测量线粒体分段的纹理属性。将输出属性标记为“线粒体纹理”。
找到核子(2)。设置通道“ DAPI”并使用方法“ C”。展开下拉菜单,然后适当调整“通用阈值”。调整的最小面积,以除去背景碎片(我们使用> 20微米2 )(图3D)。将输出总体标记为“ Nuclei(DAPI)”。
定义结果。使用方法“输出列表”。对于每个人口,展开下拉菜单,然后从每个测量的参数中选择所需的输出类型。或者,使用“全部应用”为该总体中所有测得的参数选择输出类型。单击构建基块顶部的绿色箭头以应用更改,然后保存协议。


图3.使用哥伦布图像数据存储和分析系统选择分析协议。A.使用Alexa 488通道进行“查找神经突”选择,请参阅数据分析协议的步骤4c。B.使用Alexa 488通道进行“查找图像区域”选择,请参阅步骤4h。C.使用分段的线粒体通道“选择种群”,请参阅步骤4l。缩进的图像显示了各个选择的特写。D.使用DAPI通道进行“查找核”选择,请参阅步骤4q。所有比例尺= 20μm 。



选择“批次分析”选项卡,然后从左侧面板中选择要分析的数据文件夹。然后,选择上面编写的分析协议。单击绿色箭头开始分析。
分析完成后,结果将显示在左侧面板中的相应数据文件夹中。选择浏览选项卡。通过单击旁边的加号打开相应的数据文件夹,然后选择“结果”文件以将其打开。在右下方的面板中,“摘要”选项卡将显示该分析的数据。单击与“结果”文件的名称相对应的相邻标签,以完整查看原始数据。使用蓝色的“下载”链接下载文件。
在Excel 2016中打开下载的文件。整理数据,以便按顺序对每种浓度下每种化合物以及对照孔的参数值进行排序。
使用每个参数的单独数据表,将每个化合物的数据输入到GraphPad Prism 8.2中。确保每种化合物按列组织,每种浓度按行组织。包括阳性对照化合物的数据。
统计一nalysis


统计分析将取决于实验设置。但是,应控制某些重要的板反应,以确保测定的鲁棒性和可重复性。


创建另一个数据表,并在整个板上绘制核数(DAPI)。这应保持一致,以最大程度地减少印版漂移影响。
创建另一个数据表,并针对每个参数针对DMSO鱼藤酮控制井绘制DMSO车辆控制井的值(图4A)。一个显著差异应被视为对每个参数。
计算Z'和SW分数,以确保测定的鲁棒性。公式为:










在此协议中,“最大值”是指DMSO车辆控制井中的值,而“最小值”是指DMSO鱼藤酮控制井中的值。图4B中给出了该测定中各种参数的Z′得分的示例数据。通常,在表型筛选分析中可以使用高于0.5的Z'评分和高于2的SW评分。


通常,将进行采用Dunnet事后测试的单向ANOVA,以比较单一浓度药物对溶媒处理井的作用。或者,如果进行了浓度响应板图分析,通常使用对数(激动剂)对响应(三个参数)的数据进行非线性回归(曲线拟合)分析。所有分析均在GraphPad Prism 8.2中进行。



图4.显示鱼藤酮效应的实例数据,来自GFP-LUHMES 384孔板药物筛选的Z'得分。一。通过比较DMSO媒介物处理过的孔与DMSO鱼藤酮处理过的孔的值,显示了三个示例参数的鱼藤酮效应。在这两种情况下,鱼藤酮处理过的孔的平均参数值与媒介物处理过的孔均显着不同(*** = P < 0.0001)。B.在四个分开的板上的几个参数的Z'得分,每个得分相隔三周。参数“ Neurite圆度”表示通过比较单元格面积与单元格周长来度量单元格形状;与球形细胞的分数最高为1相比,具有广泛神经突的健康神经元的分数非常低,接近0。



笔记



一旦到达第15代,GFP-LUHMES细胞应被淘汰,并以年轻的种群代替。
冷冻GFP-LUHMES储备液时,建议拆分一个完全融合的T75烧瓶,将沉淀重悬于5 ml LUHMES增殖培养基中,加入10%DMSO和20%FBS,然后在5个冷冻小管之间均分。
在血球计数器上计数GFP-LUHMES细胞时,仅应计数圆形的明亮球体。
当将已分化的GFP-LUHMES细胞转移至384孔板时,应迅速采取行动,并在两次转移之间使用多通道移液器将细胞悬液重新匀浆。这是为了确保每个孔中铺板的细胞数量在整个板上尽可能一致。
分析软件可以输出其他一些参数,包括细胞数量和带有神经突的细胞数量,这些参数对于评估细胞活力,跨板漂移和条件以及分析天数之间的变异性非常有用。
菜谱



无菌1x PBS
在适合高压灭菌的烧瓶中将5片PBS片加入500毫升蒸馏水中
混合直至片剂溶解并高压灭菌
室温保存
无菌1x胰蛋白酶
向适合高压灭菌的烧瓶中加入500毫升蒸馏水
将烧瓶高压灭菌并转移到无菌培养罩中
在通风橱中,将45 ml无菌蒸馏水加到50 ml F alcon中的5 ml胰蛋白酶10x中
颠倒2-3次进行混合
储存在4°C
LUHMES基础媒体
储存在4°C


500毫升Advanced DMEM / F-12                         
5毫升N-2补品                         
5毫升Pen-Strep                                       
                                          5毫升L-谷氨酰胺


LUHMES扩散媒体
在4°C下存放一周


50毫升LUHMES基本培养基                         
40 ng / ml碱性FGF


LUHMES差异化媒体
每次使用都准备新鲜


20 ml LUHMES基础培养基                                       
1微克/毫升四环素


1毫米dCAMP                                                       
2 ng / ml GDNF                                                     

致谢



这项工作由HM(F-1301)的帕金森英国高级研究金和HM(I-1904)的帕金森英国虚拟生物技术项目资助。LF得到医学科学院(SBF002 \ 1142)和医学研究理事会(MRC,拨款1812144)的支持。IV由墨西哥Consejo Nacional de Ciencia y Technologia (CONACyT )支持。


GFP-LUHMES细胞的产生是基于Ratcliffe等人先前建立的方案。,2018。




竞争我nterests



作者宣称没有利益冲突。



参考文献



阿姆斯特朗(MJ)和奥肯(MS)(2020)。帕金森病的诊断和治疗:综述。JAMA 323(6):548-560。              
              DELP ,J.,冯克,M.,鲁道夫,F.,Cediel ,A.,Bennekou ,SH,范德华STEL ,W.,Carta的,G.,詹宁斯,P.,托马,C.,加德纳,I. ,van de Water,B.,Forsby A.和Leist M.(2019)。开发了一种可检测线粒体有毒物质的神经毒性测定方法。弓毒93(6):1585-1608。
Dolga ,AM,de Andrade,A.,Meissner,L.,Knaus ,HG,Höllerhage ,M.,Christophersen ,P.,Zischka ,H.,Plesnila ,N.,Hoglinger ,GU和Culmsee ,C。(2014年) 。人多巴胺能神经元中SK通道的亚细胞表达和神经保护作用。细胞死亡Dis 5:e999。
豪瑟,DN和黑斯廷斯,TG(2013)。帕金森氏病和单基因帕金森症的线粒体功能障碍和氧化应激。Neurobiol Dis 51:35-42。
Höllerhage ,M.,Moebius,C.,Melms ,J.,Chiu,W.-H.,Goebel,JN,Chakroun ,T.,Koeglsperger ,T.,Oertel ,WH,Rösler ,TW,Bickle ,M.和霍格林格,顾(2017)。通过在LUHMES细胞中进行化合物筛选,揭示了磷酸二酯酶1抑制物对α-突触核蛋白毒性的保护作用。科学代表7(1):11469。
              克鲁格,AK,巴尔末,NV,马特,F.,Schonenberger ,F.,Merhof ,D。和Leist ,M.(2013)。评估人神经突生长试验作为发育性神经毒性物质的特异性筛选方法。拱毒理学87(12):2215至2231年。              
Lotharius ,J.,巴尔格,S.,Wiekop ,P.,伦德伯格,C.,雷蒙,HK和布伦丁,P。(2002)。突变型α-突触核蛋白对新型人中脑细胞系中多巴胺稳态的影响。生物化学杂志277(41):38884-38894。              
Lotharius ,J.,Falsig ,J.,面包车比克,J.,佩恩,S.,Dringen ,R.,布伦丁,P。和Leist ,M。(2005)。由多巴胺依赖性氧化应激触发的人中脑神经元衍生细胞的逐步变性取决于混合谱系激酶途径。Ĵ神经科学25(27):6329-6342。              
Park,JS,Davis,RL和Sue,CM(2018)。帕金森氏病中的线粒体功能障碍:新的机制见解和治疗观点。Curr Neurol Neurosci Rep 18(5):21。
Mortiboys ,H.,Aasly ,J.和Bandmann ,O.(2013)。熊毛酸可拯救家族性帕金森氏病常见形式的线粒体功能。脑136(Pt 10):3038-50。
山口,一。,石川,K 。我。,Inoshita ,T 。,芝福岛,K 。,Saiki ,S . ,多野,Ť 。,森,A 。,Oji ,Y 。,Okuzumi ,一。李,ÿ 。,船山,中号。,今井,Y 。,Hattori ,N 。and Akamatsu ,W.(2020)鉴定改善家族性帕金森病神经元线粒体清除障碍的治疗剂。 干细胞报告14(6):1060-1075。
              Poewe ,W.,Seppi,K.,Tanner,CM,Halliday,GM,Brundin ,P.,Volkmann,J.,Schrag ,AE和Lang,AE(2017)。帕金森综合症。Nat Rev Dis Primers 3:17013。
              拉特克利夫,LE,克斯Villaseñor ,I.,詹宁斯,L.,荒地,PR,Mortiboys ,H.,瓦尔兹特鲁,A.,Karyka ,E.,辛普森,JE,因斯,PG,加伍德,CJ和沃顿,SB( 2018)。人星形胶质细胞中IGF1R的缺失改变了复合物I的活性和对神经元的支持。神经科学390:46-59。
Scholz,D.,Pöltl ,D.,Genewsky ,A.,Weng,M.,Waldmann ,T.,Schildknecht ,S. and Leist ,M.(2011)。从人类LUHMES细胞系快速,完整和大规模生成有丝分裂后神经元。Ĵ神经化学杂志119(5):957-971。
              斯蒂格勒,内华达州,克鲁格,AK,马特·F和莱斯特·米(2011)在高密度培养物中通过多参数活细胞成像评估化学诱导的人神经突增生损伤。毒理学科学121(1):73-87。              
Tong,ZB,Hogberg ,H.,Kuo ,D.,Sakamuru ,S.,Xia,M.,Smirnova,L.,Hartung,T.和Gerhold ,D.(2017)。用于高通量神经元细胞毒性筛选的三种人类细胞系模型的表征。J Appl毒理学杂志37(2):167-180。
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引用:Leah, T., Vazquez-Villaseñor, I., Ferraiuolo, L., Wharton, S. B. and Mortiboys, H. J. (2021). A Parkinson’s Disease-relevant Mitochondrial and Neuronal Morphology High-throughput Screening Assay in LUHMES Cells. Bio-protocol 11(1): e3881. DOI: 10.21769/BioProtoc.3881.
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