We thank Prof. Kelvin Luk for his generous gift of &#945;-synuclein PFFs, Conjung Zheng for establishing and culturing neuronal cells, and the Light Microscopy Unit at the Institute of Biotechnology, University of Helsinki, for imaging immunostained cells. This work was supported by grants from 3i-Regeneration by Business Finland (Finnish Funding Agency for Innovation), Academy of Finland #309489, #293392, #319195; Sigrid Juselius Foundation, and the statutory funds of the Maj Institute of Pharmacology, PAS, Poland.

<ol>
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The authors have nothing to disclose.

Spreading Lewy pathology, of which pS129-&#945;syn is a major constituent, is a histopathological hallmark of PD. Stopping or slowing down the accumulation of aggregated pS129-&#945;syn may slow down the degeneration of dopamine neurons and the progression of alpha-synucleinopathy. However, a mechanistic understanding of how pS129-&#945;syn aggregation contributes to the demise of dopamine neurons still has to be established. Evidence from human postmortem studies on brain samples from patients at different stages of the disease as well as observation of pS129-&#945;syn positive inclusion in transplanted fetal neurons strongly suggests the spreading of Lewy pathology between cells16,17,33. Consequently, prion-like spreading of pS129-&#945;syn was recently recapitulated by using &#945;-synuclein PFFs9,10. Establishing a robust, cost-effective, and relatively high- or medium-throughput model of pS129-&#945;syn spreading and accumulation, specifically in dopamine neurons, can considerably speed up the search for novel treatments and compounds modifying this process.
Because loss of dopamine neurons is the main cause of motor symptoms in PD and these cells possess many unique properties2,34,35, modeling of prion-like spreading of pS129-&#945;syn in dopamine neurons is the most relevant type of model from the translational perspective. Protocols utilizing micro island cultures of embryonic midbrain neurons on 4 well plates and semiautomatic quantification have been described previously18. The protocol described here was adapted to 96 well plates and provides less laborious preparation of micro islands, allowing for the preparation of up to four plates containing 60 wells each by an experienced researcher during one workday. Culturing dopamine neurons in 96 well plates allows for testing drugs at lower amounts and enables high transduction rates with lentivirus vectors. It is also possible to combine different treatments to perform more complex experiments.
Before applying any treatments (including PFFs), the quality of the culturing should be checked with bright-field microscopy. If the microscopy system does not utilize a CO2 chamber with heating, the cells should not be kept outside of the incubator for more than couple of minutes, because primary mouse dopamine neurons are delicate and easily stressed. For the same reason, it is advised that the first imaging should be done after 24 h of incubation (between DIV1-DIV3). The cells should appear to be alive with present cell bodies and homogenously spread inside the micro island. Primary neurons would have settled on the PO-coated ground and started to establish neuronal projections. It is possible to observe small clump of cells (i.e., diameter smaller than 150&#8211;200 &#956;m) that can be formed if the trituration process is not done properly or plating density is higher than recommended. These small clumps would not affect the experiment, unless they are more than a few per well and/or larger. Clumped cells make it very difficult to identify immunohistochemical markers and individual cells during the image analysis. It is essential to avoid such clumps by careful coating, triturating, and controlling plating density. If the uniformity of these conditions cannot be observed at certain wells, do not include these defective wells in the experiment. Such exclusion should be done before the execution of any treatments.&#160;
Moreover, utilization of 96 well plates allows for convenient multichannel pipette use during staining procedures and direct visualization with automatic plate microscopes, further increasing throughput. Utilization of automated image quantification is indispensable for the analysis of the data from high-content imaging platforms. In addition to the capability to process thousands of images obtained from each experiment, it ensures unbiased, identical quantification of all treatment groups. The workflow proposed for the image analysis is based on simple principles of segmenting dopamine neurons, filtering correctly segmented cells by supervised machine learning, and subsequently quantification of phenotypes (pS129-&#945;syn positive and pS129-&#945;syn negative), again by supervised machine learning. Although several different approaches for this task can be envisioned, we have found the combination of segmentation with machine learning to be the most robust for dopamine cultures due to high plating density, the diverse shapes of dopamine neurons, and the presence of strongly stained neurites. The proposed image analysis algorithm was implemented in CellProfiler and CellProfiler Analyst, open source, freely available high-content image analysis software26,27. The algorithm could also be implemented with other image analysis software, either open source (e.g., ImageJ/FIJI, KNIME) or proprietary. However, in our experience these often sacrifice customization capabilities for ease of use, and therefore might not perform well in complicated analyses. We have found that the CellProfiler and CellProfiler Analyst software packages give particularly reliable results by combining a substantial number of implemented algorithms, extreme flexibility in designing workflow, and simultaneously handling and efficiently processing of high-content imaging data.
The described protocol could also be adapted for quantification of other cellular phenotypes characterized by immunostaining with different antibodies, such as markers of other neuronal populations (e.g., DAT, GAD67, 5-HT etc.) and protein aggregates (e.g., phospo-Tau, ubiquitin). Multiple fluorescent markers could also be combined to distinguish multiple phenotypes (e.g., cells with inclusions at different stages of maturation). Automated classification of multiple phenotypes should also be easy to implement in the described image analysis pipelines by merely adding a channel containing immunofluorescent images of additional markers to measurement steps and sorting cells into multiple bins. Utilization of multiple markers at the same time would, however, require the optimization of immunostaining and imaging conditions. Additionally, for better quality in immunofluorescence imaging, the use of special black-walled 96 well plates explicitly designed for the fluorescent microscope is recommended. However, these can be considerably more expensive than standard cell culture plates, which are sufficient for the analysis described in our protocol.
The type and quality of utilized PFFs are critical for the outcome of the experiments. PFFs can both affect the robustness of the assay and the interpretation of results. Preparation conditions might affect the seeding efficiency of PFFs and, indeed, PFF &#34;strains&#34; with different physiological properties have been reported36. Nonetheless, the preparation and validation of PFFs are beyond the scope of this article and have been described in several publications11,28,29,30. In addition to the preparation protocol, the species of origin of &#945;-synuclein in PFFs (e.g., mouse, human) and the usage of wild type or mutated protein (e.g., human A53T &#945;-synuclein) should be considered, depending on the particular experimental conditions. Induction of pS129-&#945;syn accumulation by PFFs was shown to be dependent on age of the culture (i.e., days in vitro), with more mature cultures showing more pronounced induction11. This is probably due to the increased number of neuronal connections in more mature cultures, and increased &#945;-synuclein protein levels. In our hands, treatment with PFFs at DIV8 gave the most robust results, with pronounced accumulation of pS129-&#945;syn in dopamine neuron soma, while not compromising neuronal survival. The described protocol is well suited to study treatments modifying early events leading to aggregation of endogenous &#945;-synuclein because we quantify pS129-&#945;syn positive inclusions at a relatively early time point, 7 days after inoculation with PFFs. At this time point, intrasomal inclusions are present in a significant fraction of cells and can be easily distinguished by immunostaining while no PFF-induced cell death is observed, simplifying the interpretation of the results. Importantly, as the morphology and composition of PFF-induced inclusions can change over time12,13, the described protocol could, in principle, be modified to study more mature inclusions by fixation and immunostaining at later time points. However, keeping dopamine neurons in culture for longer than 15 days requires extreme care, and might induce additional variation because of cells failing to survive independently from PFF inoculation. Additionally, more extended cultures complicate drug treatment schedules. Many compounds have limited or not poorly characterized stability in the cell culture medium, and replenishment of a drug is not trivial because complete exchange of medium compromises the survival of the dopamine cultures.
Phosphorylation of &#945;-synuclein at Ser129 is consistently reported in PFF-based models of &#945;-synuclein aggregation and colocalizes with markers of misfolding and aggregation such as Thioflavin S, ubiquitin, or conformation-specific antibodies11,12. In our hands, immunostaining for pS129-&#945;syn also gives the strongest signal with the lowest background and is most straightforward to analyze, giving robust results when multiple treatments are screened. Importantly, immunostaining with pS129-&#945;syn antibody does not detect PFFs that remain outside of the cells, significantly reducing the background. However, it is important to remember that Ser129 phosphorylation is probably one of the earliest processes linked with the misfolding of &#945;-synuclein and might be differently regulated under specific conditions. Therefore, any findings that show positive effects on pS129-&#945;syn should be confirmed by other markers.
Statistical analysis should be tailored correspondingly to experimental design. It is essential to perform experiments in at least three independent biological replicates (i.e., separate primary neuronal cultures). These replicates should be plated on different plates and treated independently. We analyze the data obtained from replicates on different plates with random block design ANOVA37 to take into account the pairing of data for different experimental plates.&#160;

Parkinson&#8217;s disease (PD) is a neurodegenerative disorder characterized by the death of the midbrain dopamine neurons in the substantia nigra (SN), subsequent loss of dopamine tone in basal ganglia, and consequent motor impairments1,2. A major histopathological feature in the brains of PD patients are intracellular protein/lipid aggregates found in neuronal soma, called Lewy bodies (LB), or in neurites, Lewy neurites (LN), collectively known as Lewy pathology3. Lewy pathology in the brain appears to progress with advancing PD resembling the spread of pathogenic factors through neuronal connections. Abundant Lewy pathology is found in dopamine neurons in the SN and cells in other areas affected by neurodegeneration4. However, during disease progression, spread and onset of protein aggregation do not always correlate with neuronal death and the exact contribution of Lewy pathology to neuronal death is still unclear5.
LB and LN had been shown to consist of membranous and proteinaceous components3. The former are membrane fragments, vesicular structures (possibly lysosomes and autophagosomes) and mitochondria3. The latter consists of at least 300 different proteins6. A hallmark study by Spillantini et al.7 demonstrated that the major protein component of Lewy pathology is &#945;-synuclein. Highly expressed in neurons, and linked with membrane fusion and neurotransmitter release, &#945;-synuclein in Lewy pathology is present mostly in misfolded, amyloid fibril form, the bulk of which is phosphorylated at Ser129 (pS129-&#945;syn)4,8.
Importantly, it was also demonstrated that due to its prion-like properties, misfolded &#945;-synuclein might have a causative role in Lewy pathology formation4. The prion-like properties of misfolded &#945;-synuclein were shown with both midbrain extracts from patients and exogenously prepared &#945;-synuclein preformed fibrils (PFFs) to induce &#945;-synuclein aggregates in neurons in culture and in vivo9,10. PFFs present a reliable and robust model to study the progression of &#945;-synuclein pathology in dopamine neurons. When PFFs are applied to cultured primary neurons or injected into the animal brain, they lead to the formation of &#945;-synuclein-containing inclusions in neurites and cell soma11 that recapitulate many features seen in Lewy pathology. Observed inclusions are detergent-insoluble in Triton X, ubiquitinated, stained with the amyloid specific dye Thioflavin S, and contain &#945;-synuclein hyperphosphorylated at Ser12911,12. Importantly, these inclusions do not form in &#945;-synuclein knockout animals11, indicating the dependence of their formation on endogenous &#945;-synuclein.
Nonetheless, it is difficult to directly compare PFF-induced inclusions and Lewy pathology found in PD patients because human LBs and LNs are highly heterogeneous3. Observed heterogeneity of Lewy pathology might be caused by different stages of the formation, different anatomical location, or differences in the conformation of misfolded &#945;-synuclein initiating the aggregation process. The same factors might influence PFF-induced pS129-&#945;syn positive inclusions. Indeed, recently it was demonstrated that PFF-induced pS129-&#945;syn positive inclusions in primary neuronal cultures represent very early stages of pathology that can mature to structures closely resembling LB after prolonged incubation period12,13.
Modeling early spreading and accumulation of misfolded &#945;-synuclein with PFFs is valuable for drug development, as Lewy pathology spread is considered one of the early-stage disease markers. Therefore, aggregation-preventive treatments may be promising for stopping or slowing down the progression of PD at very early stages. Several clinical trials aimed at slowing or stopping &#945;-synuclein accumulation are ongoing14. For later-stage patients, transplantation of dopamine neuronal progenitors can be a better treatment alternative15. However, Lewy pathology was documented in transplanted embryonic neurons during the post-mortem analysis of PD patient brains16,17, also indicating the need for protection against &#945;-synuclein accumulation.
In vitro, &#945;-synuclein PFFs are known to induce aggregation in immortalized cell lines, or more commonly, in rodent primary hippocampal or cortical neurons. Neither of these are close to recapitulating dopamine neurons10. Culturing these neurons requires dense plating of certain numbers of neurons in vitro18. To achieve high plating density with limited material (e.g., primary dopamine neurons), the micro island culturing method is commonly utilized. In micro island culturing, cells are initially plated in a small drop of medium (usually a few microliters) kept together by surface tension in the middle of a large well18. After the neurons attach, the entire well is filled with the medium while the cells remain confined at high density in the small plating area. In addition to achieving high plating density, micro islands also prevent plating near the edges of wells, where variations in cell density and survival are frequent. Micro islands are often utilized in relatively large wells or dishes; however, establishing midbrain neuronal cultures in micro islands in 96 well plate format enables the study of Lewy pathology in bona fide dopamine neurons with medium-to-high-throughput power. In vitro experiments with these neurons allowed us to discover the glial cell line-derived neurotrophic factor (GDNF), which promotes survival of mature dopamine neurons in vitro and in vivo19,20,21,22 and also prevents the formation of &#945;-synuclein aggregates in dopamine neurons23. Human patient-induced pluripotent stem cell-derived dopamine neurons constitute a more accurate model due to their human origin and longer survival time in vitro. However, induction of &#945;-synuclein pathology in human neurons is observed after multiple months, compared to a week in mouse embryonic neurons, and/or with multiple stressors (e.g., combination of &#945;-synuclein overexpression and PFFs)24,25. In addition, maintenance of human dopamine neurons is more costly and laborious when compared to primary embryonic neurons, essentially limiting their use in high-throughput applications.
Further, primary dopamine neuronal cultures can be genetically modified (e.g., with CRISPR/Cas9) and/or treated with pharmacological agents23. They constitute a fast and reproducible platform for applications like molecular pathway dissection and drug library screening. Even though limited material can be obtained from these cultures, it is still possible to conduct small size genomics/proteomics analyses. Culturing primary neurons in 96 well format is better for immunocytochemistry and fluorescence microscopy techniques, followed by high-content phenotype analysis. Multispectral fluorescence images derived from automated imaging of 96 well plates can be converted into quantitative results (e.g., the number of LB-containing neurons per well). Such analyses can be done with free software, such as CellProfiler26,27. Overall, primary embryonic midbrain cultures plated in 96 well plates provide a robust and efficient platform to study dopamine neurons and &#945;-synuclein aggregation with the opportunity for high-throughput phenotype screening.

<table><tbody><tr><td>0.4% Trypan Blue stain</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>15250061</td><td></td></tr><tr><td>15 ml CELLSTAR Polypropylene tube</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td>188261</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>10236276001</td><td></td></tr><tr><td>5 M HCl</td><td>N/A</td><td>N/A</td><td>Media kitchen, Institute of Biotechnology, University of Helsinki</td></tr><tr><td>5 M NaOH</td><td>N/A</td><td>N/A</td><td>Media kitchen, Institute of Biotechnology, University of Helsinki</td></tr><tr><td>96-well ViewPlate (Black)</td><td>PerkinElmer, Waltham, Massachusetts, USA</td><td>6005182</td><td></td></tr><tr><td>99.5% Ethanol (EtOH)</td><td>Altia Oyj, Rajam&amp;auml;ki, Finland</td><td>N/A</td><td></td></tr><tr><td>AquaSil Siliconizing Fluid</td><td>Thermo Scientific, Waltham, Massachusetts, USA</td><td>TS-42799</td><td></td></tr><tr><td>Autoclaved 1.5 ml microcentrifuge tube</td><td>N/A</td><td>N/A</td><td>Media kitchen, Institute of Biotechnology, University of Helsinki</td></tr><tr><td>Bioruptor sonication device</td><td>Diagenode, Liege, Belgium</td><td>B01020001</td><td></td></tr><tr><td>Ca2+, Mg2+ free Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>14175-053</td><td></td></tr><tr><td>CellProfiler and CellAnalyst software packages</td><td>N/A</td><td>N/A</td><td>free open-source software</td></tr><tr><td>Centrifuge 5702</td><td>Eppendorf AG, Hamburg, Germany</td><td>5702000019</td><td></td></tr><tr><td>CO2 Incubator</td><td>HeraCell, Thermo Scientific, Waltham, Massachusetts, USA</td><td>N/A</td><td></td></tr><tr><td>Counting chamber</td><td>BioRad Inc., Hercules, California, USA</td><td>1450015</td><td></td></tr><tr><td>DeltaVision Ultra High Resolution Microscope with air table and cabinet</td><td>GE Healthcare Life Sciences, Boston, Massachusetts, USA</td><td>29254706</td><td></td></tr><tr><td>Deoxyribonuclease-I (Dnase I)</td><td>Roche, Basel, Switzerland</td><td>22098700</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>G8769</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>21331&amp;ndash;020</td><td></td></tr><tr><td>donkey anti-mouse AlexaFluor 488</td><td>Thermo Scientific, Waltham, Massachusetts, USA</td><td>A21202</td><td></td></tr><tr><td>donkey anti-rabbit AlexaFluor 647</td><td>Thermo Scientific, Waltham, Massachusetts, USA</td><td>A31573</td><td></td></tr><tr><td>donkey anti-sheep AlexaFluor 488</td><td>Thermo Scientific, Waltham, Massachusetts, USA</td><td>A11015</td><td></td></tr><tr><td>Dulbecco&#039;s buffer</td><td>N/A</td><td>N/A</td><td>Media kitchen, Institute of Biotechnology, University of Helsinki</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>10500056</td><td></td></tr><tr><td>Fisherbrand Plain Economy PTFE Stir Bar</td><td>fisher scientific, Thermo Scientific, Waltham, Massachusetts, USA</td><td>11507582</td><td></td></tr><tr><td>ImageXpress Nano Automated Imaging System</td><td>Molecular Devices, San Jose, California, USA</td><td>N/A</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>25030&amp;ndash;032</td><td></td></tr><tr><td>mouse monoclonal anti-tyrosine hydroxylase</td><td>Millipore, Merck KGaA, Darmstadt, German</td><td>MAB318</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td><td>17502&amp;ndash;048</td><td></td></tr><tr><td>Normal Horse Serum</td><td>Vector Laboratories Inc., Burlingame, California, United States</td><td>S-2000</td><td></td></tr><tr><td>paraformaldehyde (PFA)</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>158127</td><td></td></tr><tr><td>paraformaldehyde powder, 95%</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>158127</td><td></td></tr><tr><td>pH-Fix, color-fixed indicator strips</td><td>Macherey-Nagel, D&amp;uuml;ren, Germany</td><td>92110</td><td></td></tr><tr><td>Phospohate Buffer Saline (PBS)</td><td>N/A</td><td>N/A</td><td>Media kitchen, Institute of Biotechnology, University of Helsinki</td></tr><tr><td>poly-L-ornithine</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>P4957</td><td></td></tr><tr><td>Primocin</td><td>InvivoGen, San Diego, California, USA</td><td>ant-pm-1, ant-pm-2</td><td></td></tr><tr><td>rabbit monoclonal anti-alpha-synuclein filament</td><td>Abcam, Cambridge, United Kingdom</td><td>ab209538</td><td></td></tr><tr><td>rabbit monoclonal anti-phospho-serine129-alpha-synuclein</td><td>Abcam, Cambridge, United Kingdom</td><td>ab51253</td><td></td></tr><tr><td>RCT Basic, IKA Magnetic Stirrer</td><td>IKA&amp;reg;-Werke GmbH, Staufen, Germany</td><td>3810000</td><td></td></tr><tr><td>recombinant human glia-derived neurotrophic factor (hGDNF)</td><td>PeproTech, Rocky Hill, NJ, USA or Prospec, Ness-Ziona, Israel</td><td>450-10 (PeproTech), CYT-305 (Prospec)</td><td></td></tr><tr><td>recombinant mouse alpha synuclein Pre-Formed Fibrils (PFFs)</td><td>N/A</td><td>N/A</td><td>Gift from collaborator, Prof. Kelvin C Luk</td></tr><tr><td>Research Stereomicroscope System</td><td>Olympus Corporation, Tokyo, Japan</td><td>SZX10</td><td></td></tr><tr><td>sheep polyclonal anti-tyrosine hydroxylase</td><td>Millipore, Merck KGaA, Darmstadt, German</td><td>ab1542</td><td></td></tr><tr><td>TC 20 Automated Cell Counter</td><td>BioRad Inc., Hercules, California, USA</td><td>1450102</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>11332481001</td><td></td></tr><tr><td>trypsin</td><td>MP Biomedicals, Valiant Co., Yantai, Shandong, China</td><td>2199700</td><td></td></tr></tbody></table>

studying pre-formed fibril induced &#945;-synuclein accumulation in primary embryonic mouse midbrain dopamine neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

A few days after the plating (DIV1&#8211;DIV3), bright-field microscopy was done to assess the health and homogenous spread of the cultured cells, and uniformity of these conditions at the individual wells (Figure 3). Cultured midbrain cells were spread homogenously within the micro island created before the plating (Figure 3A,B). Primary neurons had settled on the coated ground homogenously and established neuronal projections (Figure 3B). A small clump of cells (diameter smaller than 150 &#956;m) was observed at the well and shown as an example (Figure 3C).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61118/61118fig03.jpg" /> 
Figure 3: A few days after the plating (e.g., DIV3), the condition of primary midbrain cultures were observed with bright-field microscopy. (A) Cultured cells spread across the middle of the well within the PO-coated micro island with an approximate radius of 4.4 mm (shown with white arrows) created by scratching PO from approximately 1 mm well perimeter (shown with black arrows). (B) Cultured primary neurons homogenously spread within the area and neuronal projections (marked with red arrowheads) were observed. (C) A cell clump with a diameter smaller than 150 &#956;m is marked with blue arrowheads. Scale bars = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61118/61118fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Primary mouse midbrain cultures were immunostained with anti-TH and anti-pS129-&#945;syn antibodies and imaged with an automated microscope after 15 days in vitro. Coated micro islands provided restricted area for the attachment of cells in the middle of wells (Figure 4A,B). Dopamine neurons immunolabeled with TH marker were spread around the micro island in a monolayer, separated from each other, without any clumping (Figure 4A&#8217; and 4B&#8217;).
<img alt="Figure 4" class="xfigimg bigfig" src="/files/ftp_upload/61118/61118fig4v2.jpg" /> 
Figure 4: At DIV15, 800&#8211;1,000 dopamine cells were quantified from each micro island, with or without PFF treatment. Representative images of embryonic midbrain cultures immonustained with anti-TH and anti-pS129-&#945;syn antibodies. (A, A&#8217;, A&#8217;&#8217;) Control cells without PFF treatment. (B, B&#8217;, B&#8217;&#8217;) PFF-treated cells with pS129-&#945;syn inclusions. (C) Quantification of TH-positive cell numbers in wells treated with vehicle (control), PFFs, or PFFs with 50 ng/mL GDNF. (D) Quantification of pS129-&#945;syn aggregates in TH-positive cells treated with vehicle (control), PFFs, or PFFs with 50 ng/mL GDNF. Statistical significance was calculated with random block design ANOVA. **p &#60; 0.01, n = 4 individual plates (biological replicates), each with 3&#8211;6 wells per treatment group (technical repeats). Scale bars = 300 &#956;m for A, B; 50 &#956;m for A&#8217;, B&#8217;; 25 &#956;m for A&#8217;&#8217;, B&#8217;&#8217;. <a href="https://www.jove.com/files/ftp_upload/61118/61118fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
While cultures without PFF treatment did not have any pS129- &#945;syn signal (Figure 4A&#8217; and 4A&#8217;&#8217;), cultures treated with &#945;-synuclein PFFs developed pS129-&#945;syn positive inclusions (Figure 4B&#8217; and 4B&#8217;&#8217;). In vitro PFF treatment for 7 days did not cause any significant decrease in numbers of TH-positive neurons, compared to other experimental groups (Figure 4C). PFF-treated cultures had a population of ~40% of pS129-&#945;syn positive TH-positive dopamine neurons. Treatment with positive control, GDNF, reduced the percentage of TH-positive dopamine neurons with pS129-&#945;syn positive inclusions (Figure 4D, see also the raw data and example images in the Supplementary Files).
Supplementary Files: Example pipelines for high-content image analysis with CellProfiler and the CellAnalyst software packages, example images, and raw data for Figure 4E, F. (1-9) Example_Images. Open these images with ImageJ or CellProfiler. (10) Fig_4_raw_data_Er_et_al.xlxs. (11) TH_LB_V1.cpipe (Steps 6.2&#8211;6.8), (12) TH_LB_V2.cpipe (Steps 6.11&#8211;6.18)&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/61118/Supplementary_Files.zip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Studying Pre-formed Fibril Induced Î±-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons at JoVE.com

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

studying-pre-formed-fibril-induced-synuclein-accumulation-primary

Research

JoVE Journal

Neuroscience

10.5K Views.  University of Helsinki. Dit protocol biedt een robuust, reproduceerbaar model van alfa synuclein accumulatie in primaire dopamine neuronen voor het bestuderen van de mechanismen en behandelingen die deze accumulatie kunnen reguleren. Voorgevormde alfa-synucleinfibrils induceren progressieve, zeer reproduceerbare eiwitaccumulatie in combinatie met geautomatiseerde beeldvorming en onbevooroordeelde beeldanalyse, dit protocol vergemakkelijkt een relatief snelle, gemiddelde tot hoge doorvoerscreening van alfa-synucleina...

Video: Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Medicine

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

&#945;-Synuclein Propagation: Intranasal Administration in Parkinson&#39;s Disease

Technique for Intranasal Administration of &#945;-Synuclein Aggregates

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein (&#945;-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of &#945;-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of &#945;-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of &#945;-Syn aggregates in a mouse model induced &#945;-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of &#945;-Syn aggregates that induced &#945;-Syn pathology in the OB of mice. Intranasal administration of &#945;-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of &#945;-Syn pathology in the OB and the pathway of &#945;-Syn propagation through the olfactory system.

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein ...

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

Source: Breid, S. et al. <a href="https://app.jove.com/v/55503">Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.</a> J. Vis. Exp. (2017).
This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...

Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

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