This protocol provides a robust, reproducible model of alpha synuclein accumulation in primary dopamine neurons for studying the mechanisms and treatments that may regulate this accumulation. Preformed alpha-synuclein fibrils induce progressive, highly reproducible protein accumulation combined with automated imaging and unbiased image analysis, this protocol facilitates a relatively fast, medium to high throughput screening of alpha-synuclein accumulation inhibiting trucks. Progressive alpha-synuclein accumulation may be one of the factors compromising neuronal function in Parkinson's disease and certain nuclear bodies.
New molecules blocking such accumulation may become new therapies for neuro degeneration. We believe that this protocol is an important step for establishing a standard and reliable tool to study molecular mechanisms regulating alpha-synuclein accumulation in Dopamine neurons. Demonstrating the procedure will be Julia Konovalova, a PhD student from my laboratory.
Before setting up the primary embryonic midbrain cell culture, add 50 microliters of dopamine, the neuron medium, to each well of the Poly-L-Ornithine coated 96 well plate and use a 100 microliter pipette tip to aspirate the medium while simultaneously scratching the bottom of each well in a circular direction to remove the coating at the perimeter of each well. A Poly-L-Ornithine coated island will remain in the middle of each well. Add 10 microliters of medium to the middle of each coated island to create micro islands.
To set up primary embryonic midbrain cultures from mouse embryonic day 13.5 mouse embryos. Pool all of the harvested midbrain floors into the same 1.5 milliliter tube and wash the samples three times with 500 microliters of calcium and magnesium free HBSS per wash. After the last wash replace the HBSS with 0.5%trypsin for a 30 minute incubation at 37 degrees Celsius.
At the end of the incubation add 500 microliters of a freshly prepared DNase I in FBS solution to the partially digested tissue and use a siliconized glass pipette with a fire polished tip to triturate the tissue. When only tiny, barely visible particles can be observed allow these tissue fragments to settle to the bottom of the micro centrifuge tube and transfer this supernatant into an empty 15 milliliter conical polypropylene tube. Add one milliliter of HBSS to the tube containing DNase I in FBS and mix several times by pipetting.
Transfer one milliliter of this solution to the remaining tissue particles and triturate the samples again. Then pull the digested cell suspension with the tube of supernatant without transferring any remaining tissue fragments after triturating the tissue sample one more time with the remaining DNase I and FBS in HBSS sediment the collected cells by centrifugation and aspirate the supernatant without disturbing the pellet. Wash the cells two times in two milliliters of warmed dopamine neuron medium per wash.
And resuspend the cells at a three times 10 to the force cells per six microliters of fresh warm, medium concentration in a micro centrifuge tube. Next remove the medium from each previously prepared micro island and mix the cells with gentle pipetting before using a 10 microliter pipette to add six microliters of cells to each micro island. When all of the cells have been added, fill the empty wells at the edges of the plate with 150 microliters of water or PBS and place the plate in the cell culture incubator for one hour.
At the end of the incubation add 100 microliters of fresh dopamine neuron medium to each well and return the plate to the incubator. On day eight of cell culture in a laminar flow hood add at 3.75 microliters of 100 microgram per milliliter of preformED fibrils to each experimental well, and 3.75 microliters of PBS to each control well. When working with PFS be cautious about unwanted protein contamination and afterwards clean the hood and all PF associated instruments with 1%SDS and 70%ethanol.
At the appropriate experimental endpoint after staining for the markers of interest, load the 96 well culture plate onto a high content plate scanner fitted with a 10 X objective. Adjust the settings according to the specifications of the 96 well plate such as the play type, manufacturer, size, distance between the wells, and the type and volume of medium. Select the imaging area of the wells to cover all of the cells in each micro island and use one well to adjust the auto focus on the DAPI expression.
Calibrate the acquisition time for each fluorescent channel based on the intensity of the staining in the control wells and adjust the parameters so that the dopamine cells in the preformed fibril treated control wells harboring phosphoserine 129 alpha synuclein aggregates within the cell soma clearly be distinguished allowing an unambiguous quantification of the phosphoserine 129 alpha synuclein positive and negative cells. Then image all of the selected wells simultaneously in all of the channels using exactly the same parameters for each well. For the analysis of the images, open CellProfiler analyst and select the V2_THpos.
properties file. To sort segmented cells into phosphoserine 129 alpha synuclein positive and negative cells first set the number of fetch cells to 50 random cells and click fetch to load images of the segmented cells. Drag at least 30 cells into the corresponding bin at the bottom of the window.
select use fast gentle boosting with 50 max rules or random forest classifiers and click train. Set fetch to 50 positive cells and click fetch to acquire example positive phosphoserine 129 alpha synuclein cells scored according to the train classifier or set fetch to 50 negative cells and click fetch to acquire example negative phosphoserine 129 alpha synuclein cells according to the train classifier. When the results are satisfactory, click score all to generate a results table summarizing the number of phosphoserine 129 alpha synuclein positive and negative dopamine neurons in each well.
A few days after plating a homogeneously spread culture can be observed by light microscopy within the micro island created before the plating. Primary neurons settle on the coated plate bottom homogeneously and establish neuronal projections. In this image, a small clump of cells less than 150 micro meters in diameter can be observed.
Immuno staining of control untreated primary mouse mid-brain cultures with neuronal cell markers 15 days after plating reveals a restricted attachment of the cells within the micro islands in the middle of the plate wells. Dopamine neurons immuno labeled with tyrosine hydroxylase marker spread around the micro island in a monolayer separated from each other without any clumping. In alpha-synuclein preformed fibril treated cultures, pS129 alpha synuclein positive inclusions can also be observed.
Treatment with in vitro preformed fibrils for seven days does not cause a significant decrease in tyrosine hydroxylase positive neuron numbers compared to other experimental groups. Treatment with glial cell line derived neurotrophic factor however, reduces the percentage of pS129 alpha-synuclein positive inclusion, harboring tyrosine hydroxylase positive dopamine neurons. Before creating micro islands, make sure that the Poly-L ornithine coated wells are solidly washed.
Also be sure to use fresh media when plating new brain cultures. The method can be combined with gene editing and pharmacological inhibitors to study the effects of specific genes and thought waves on nucleon aggregation. We routinely use this method to screen for new molecules that inhibits alpha synuclein accumulation.
Have demonstrated the accumulation protected effects of TDN and currently analyzing several small candidate molecules.
