Introduction. Approach and Methods. NeuroPrime assay for primary neurons and astrocytes

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1 Measuring Neurite Dynamics in Co-culture Using IncuCyte ZOOM Livecontent Imaging Platform and NeuroLight Red Fluorescent Label Jeremiah rown, Tricia Garay, Susana L. Alcantara, Lauren McGillicuddy, Nevine Holtz, John Rauch, Dyke McEwen, Vince Groppi, Tim Dale and Owen McManus Essen ioscience Ann Arbor, Michigan, USA & Welwyn Garden City, Hertfordshire, UK Introduction Neurite dynamics play a fundamental role in the development and function of the nervous system. Formation and maintenance of synaptic networks are necessary for nervous system function and plasticity through continuous changes in the fine structure of neurons. Neurite dynamics can be altered in disease states or following exposure to neurotoxic agents providing a rationale for understanding and quantifying this process (Conforti et al., 27). An ideal method to track neurite dynamics would allow continuous automated measurement of structural parameters, including length and number of branch points, in a non-perturbing manner. We have previously developed a quantitative mono-culture approach for investigating changes in neurite length and branching using phase images taken with an IncuCyte ZOOM live-content imaging platform in conjunction with NeuroTrack analysis software. However, in co-cultures containing neurons and astrocytes, neuronal processes cannot be unambiguously identified and measured using phase images alone. As such, we have developed a fluorescent reagent (NeuroLight Red) that specifically labels neurons in co-culture with astrocytes (Figure 1). Utilising a VSV-G pseudotyped lentivirus that encodes a red fluorescent protein (mkate2) driven off a synapsin promoter, neuronal expression is strengthened and non-neuronal crossover is minimized. Here we describe assay methods using this fluorescent label, alongside NeuroTrack algorithms, to quantitatively assess changes in neurite morphology over time in either rat primary or human ipsc-derived neuron/astrocyte co-cultures. Approach and Methods NeuroPrime assay for primary neurons and astrocytes Figure 2 provides an overview of the optimized NeuroPrime Cell Kit protocol utilizing rat primary forebrain neurons in coculture with rat primary astrocytes to measure changes in neurite length over time. Rat forebrain neurons (CellPlayer NeuroPrime rforebrain Neurons) were thawed into Neurobasal media supplemented with 27 and GlutaMax and Figure 1. Visualization of rat primary forebrain neurons using NeuroLight reagent in co-culture with astrocytes. (A) Fluorescent image of neurons at 7 days post-infection. () Merged phase and fluorescent images. Scale bar= 1µm. seeded in a 96-well poly-d-lysine coated micro-titer plate (TPP, #TPP9296) at a density of 15K cells/well. The microtiter plate was placed in an incubator for 2-4 h to allow the neurons to adhere. Neurons were then infected with NeuroLight Red, exposing the cells to lentivirus (MOI 1) for h. After the incubation period, the lentivirus was removed and media replaced.

2 2 CellPlayer 96-Well Kinetic NeuroTrack Co-culture Assay Day Day (+4 hours) Day 1 Day 3 Day 6, 9, 12, Plate rneurons Add NeuroLight Red Lentivirus 95% media replacement Plate rastrocytes egin IncuCyte scanning 5% media replacement Add Uridine + 5- FDUridine Compound Addition Figure 2. Overview of protocol for preparing neuron/astrocyte co-culture using NeuroPrime Cell Kit. 5% media replacement day 6 and beyond Rat astrocytes (CellPlayer NeuroPrime rastrocytes) were thawed into DMEM media supplemented with 15% FS and seeded at 15K cells/well on top of the rat forebrain neurons. The micro-titer plate was placed in an IncuCyte ZOOM livecontent imaging instrument, where phase and fluorescent images (4 images per well, 2x) were captured every 6 h for the entire assay duration. On day 3, 5% of the media was replaced with media containing 5-fluoro-2 -deoxyuridine and uridine (8 µg/ml and 28 µg/ml, respectively) to inhibit further astrocyte proliferation. If desired, any test samples were added at this time. Subsequently, 5% of the media volume was removed and replaced with media containing samples on every third day. Treatments and compound additions were added at two times final concentration in the media replacements. into a 96-well plate pre-coated with Matrigel, as per supplier s instructions (CDI, icell Astrocytes Prototype User s Guide). Following the infection period, the icell Neurons were dissociated and seeded on top of the icell Astrocytes. The micro-titer plate was placed in an IncuCyte ZOOM livecontent imaging platform and phase and fluorescent images (2x) were captured every 6 h for the duration of the assay. On day 3, 5% of the media was replaced with media containing 5-fluoro-2 -deoxyuridine and uridine (8 µg/ml and 28 µg/ml, respectively). Subsequently, 5% of the media volume was removed and replaced with media or media containing compounds at two times final concentration on every third day. Co-culture assay for ipsc-derived neurons and astrocytes Human induced pluripotent stem cell- (ipsc) derived neurons were supplied by Cellular Dynamics International (CDI) and prepared as described in the icell Neuron User Guide. icell Neurons or icell DopaNeurons were seeded at 3K cells/well into a 96-well TPP micro-plate pre-coated with poly-d-lysine and laminin. Neurons were infected with NeuroLight Red, exposing the cells to lentivirus (MOI 1) for h. After the incubation period, the lentivirus was removed and media replaced with icell Neuron Complete Maintenance Media. The dual species co-culture method of icell Neurons with rat primary astrocytes was comparable to that described for the CellPlayer NeuroPrime Cell Kit. However, for the single species co-culture experiments with human ipsc-derived astrocytes (icell Astrocytes), icell Neurons were infected in a T25 flask and the icell Astrocytes astrocytes were first seeded Figure 3. Segmentation masking of rat forebrain neurons. (A) Fluorescent image of neurons was obtained after 7 days in vitro. () Cell body cluster mask (blue) was applied to image. (C) Neurite mask (yellow) and both cell body cluster mask and neurite mask (D) are shown. Scale bar = 2µm

3 CellPlayer 96-Well Kinetic NeuroTrack Assay 3 Data Quantification and Analysis NeuroTrack software analyzes fluorescent images of neurons (Figure 3A) in two steps: 1) Cell body identification - Cell bodies are segmented from background based on texture and/or brightness, and are masked as cell body clusters (Figure 3). The number of clusters and the total area of clusters are normalized to image area, enabling comparison across instruments and objectives. 2) Neurite identification - Linear features are detected based on width and brightness, and are masked as neurites (Figure 3C). The total neurite length and the total number of branch points are normalized to image area. The segmentation mask can be refined and optimized for specific cell types (see NeuroTrack Co-culture Technical Note). For every timepoint of a NeuroTrack assay, the following list of metrics are generated from the automated software analysis. wells. Low intra-plate variation was observed with a standard deviation of less than 9.2 mm/mm 2 at each time-point (Figure 4). The Coefficient of Variation (%) measured in individual experiments at twelve days after plating ranged from 1.4% to 12.6% with an overall mean value of 4.4%. Further, interplate variation in maximum neurite length was assessed for control wells from 13 plates run over a 5 month period (Figure 4C). The cross plate variability for all experiments was within 2% of the overall mean neurite length. Heat maps of individual experiments revealed no significant positional effects (data not shown). The Neurotrack co-culture assay as described using the NeuroPrime Cell Kit provides a robust, medium throughput measurment of neurite dyanmics over at least a 12 day period. A Neurite length Total neurite length/mm 2 Neurite length/cell body cluster count Neurite length/cell body cluster area Cell body clusters Count/mm 2 Area/mm 2 NeuroTrack TM Metrics ranch points Total branch points/mm 2 ranch points/cell body cluster count Nucleus count (for cells with a nuclear fluorescent label) Nucleus count/mm 2 Neurite length/nucleus count ranch points/nucleus count These metrics quantify biologically relevant processes such as neurite extension, branching, and loss of neurite length due to retraction or fragementation. NeuroTrack metrics can be normalized to a fluorescent nuclear count by infecting neurons with a fluorescent nuclear marker having different spectral properties than the neurite label (green nuclear label if using NeuroLight Red for neurites). Statistical data such as standard deviation and standard error of the mean are automatically produced for user-defined replicates. Results and Discussion Assay Variability and Validation NeuroLight Red is a lentiviral reagent that has been specifically designed to efficiently transduce multiple neuronal cell types with low toxicity. Cell handling protocols and assay conditions were optimized to produce a robust 96- well plate assay capable of supporting medium throughput screening activities and mechanistic studies Assay Plate Number Figure 4. Intra- and inter-assay variability. (A) 96-well plate view displays neurite length from a rat forebrain neuron and rat cortical astrocyte co-culture over a twelve day assay. () Neurite lengths (mean ± SD) are plotted for all 96 wells from the experiment shown in panel A. (C) Neurite length was calculated in untreated control wells at 12 days after plating (mean ± SD, n=8) for 13 separate plates run over 5 months. Solid line represents overall mean neurite length ( mm/mm 2 ) and dashed lines represent the + 2% limits from the overall mean C Figure 4A displays a whole plate view of a single 96-well plate measuring neurite length over 12 days. Examination of the assay time course shows consistent responses across all

4 4 CellPlayer 96-Well Kinetic NeuroTrack Co-culture Assay NeuroLight Red infection does not significantly alter neurite length determined from NeuroLight Red images achieved maximal values at MOI 1.1 to MOI 3.3. Under these conditions, neurite length measured at day 7 using NeuroLight Red was 5-7% of the total length measured using ßIII antibodies. This is consistant with qualitative estimates comparing phase and red images, where 6-8% of neurons appear infected. Quantitative pharmacology with NeuroPrime Cell Kit: glutamate toxicity Figure 5. NeuroLight Red does not alter neurite dynamics. Fluorescent images of ßIII tubulin-stained rat forebrain neurons are shown in the absence (A) and presence () of NeuroLight Red (MOI 1.1). (C) Neurotrack software was used to calculate neurite length (mean + SD, n=4) of ßIII tubulin stained (green symbols) and NeuroLight Red expressing (red symbols) neurites from the same wells at 7 days following infection. When developing an in vitro assay using a fluorescent protein, it is important to show that the expressed protein does not affect the global biology of the system. To this end, we examined the effects of NeuroLight Red on neurite dynamics in the NeuroPrime Cell Kit using an antibody to βiii tubulin to assess total neurite length in cultures exposed to varying NeuroLight Red MOI (Figure 5). Quantification of ßIII tubulin antibody-stained fluorescent images using NeuroTrack software provides an independent measure of neurite length and can expose possible adverse effects of NeuroLight Red. The square symbols in Figure 5C represent neurite length calculated at day 7 post plating following exposure to control solutions (no NeuroLight Red lentivirus). NeuroLight Red produced no significant changes in total neurite length up to MOI 3.3 (Figure 5C, green circles). For example, at MOI 1.1, the total neurite length calculated using βiii tubulin antibody stained images was mm/mm 2 compared with 25.6 mm/mm 2 for control wells not exposed to NeuroLight Red. At MOI 1, however, neurite length declined 26.9% compared with untreated cells. This indicated that MOI less than 3 does not affect neurite development. Neurites from the same cell population were measured using NeuroLight Red (Figure 5C, red circles) prior to ßIII tubulin staining to determine the optimal MOI for investigating changes in neurite dynamics and to confirm viral expression of the fluorescent protein in these studies. Neurite length Neurite length can be a sensitive marker of neurodegenerative disease and neurotoxicity (Conforti et al., 27). Excess glutamate induced toxicity can play a role in some forms of neurotoxicity and disease progression (Choi, 1988; Mattson, 28). Investigations of glutamate neurotoxicity and other forms of neurotoxicity are often pursued using in vivo models affording limited throughput or in cellular models using short term readouts that may not easily detect chronic toxicity. The NeuroPrime Cell Kit used in conjuction with NeuroTrack software offers a long term (days to weeks) kinetic assay to quantitatively characterize potential neurotoxic effects on neurite length A C Vehicle 333 M 111 M 37 M 12 M 4 M 1.4 M Vehicle 25 µm Glutamate + 1nM MK nm MK nM MK pM MK Log [Glutamate] (M) Figure 6. Glutamate effects on neurite length. Time-course of effects of glutamate addition at day 9 (arrow) on neurite length is shown in A (mean + SEM, n=4). () Concentration-response analysis for data in A (mean + SEM, n=4) taken at 286 h time-point. (C) Addition of increasing concentrations of MK-81 1 minutes prior to addition of 25 μm glutamate (arrow) protects neurites from glutamate toxicity (mean + SEM, n=4). (D) Concentration-response data for MK-81 effects measured at 298 h (mean + SEM, n=4). Addition of glutamate to co-cultures of primary neurons and astrocytes at day 9 produced a concentration- and timedependent decrease in neurite length (Figure 6A). Measurements of neurite length at 286 hours after plating were fitted with a Hill equation, yielding a 29 μm IC 5 for glutamate-induced decreases in neurite length with a 73% D IC 5 29 M IC 5.44 nm Log [MK81] (M)

5 CellPlayer 96-Well Kinetic NeuroTrack Assay 5 decline at maximal glutamate concentrations (Figure 6). To investigate a potential mechanism mediating this toxicity, the neuron-astrocyte cocultures were exposed on day 9 to MK- 81, a specific NMDA receptor antagonist (Huettner and ean, 1988; Wong et al., 1986), 1 minutes prior to addition of a maximally effective glutamate concentration (25 μm). MK-81 afforded full protection from the effects of glutamate on neurite length (Figure 6C) with an IC 5 value of.44 nm at 298 hours (Figure 6D), suggesting that the effects of glutamate on neurite length are mediated, at least in part, by NMDA receptors These data illustrate the ability of this assay to provide quantitative pharmacology using a phenotypic assay for neurite dynamics. using more physiologically relevant cell types. In vivo studies have identified changes in neurite morphology linked to neurodegenerative diseases providing motivaton to track neurite properties in ipsc-based models of these diseasaes. For example, in Parkinson s (PD) disease models, Li et al (Li et al., 29) showed that axonal degeneration plays a crucial role in the pathogenesis of PD using an in vivo rat model. Expression of Parkinson s associated alleles produces reduced dendriditic arborization and retracted neurites in mouse models (Skibinski et al., 214; Winner et al., 211). We demonstrate in Figure 7 that the NeuroLight Red reagent successfully infects human ipsc-derived neurons (icell Neurons) and can enable quantification of neurite dynamics in a co-culture environment. While the absolute neurite length varied, the time-course of neurite development was similar for icell Neurons in co-culture with either ipsc-derived astrocytes (icell Astrocytes; Figure 7E) or rat primary astrocytes (Figure 7F). Normalized neurite length 2 A A M 125 M 32 M 8 M 2 M.5 M.1 M Vehicle 3 Figure 8. 6-OHDA induced neurotoxicity. Dopaminergic neurons (icell DopaNeurons) were labelled with NeuroLight Red, treated with increasing concentrations of 6-OHDA, and monitored for 12 days. (A) Time-course of changes in neurite length. Treaments were applied shortly before the reading taken at 114 h. Neurite length was normalized to the value at 18h (% of neurite length at 18h). () Concentration response data for changes in neurite length expressed as area under the curve (AUC) for the time period h. Data in Figure 8 were fit with a Hill equation yielding an IC 5 value of 34 µm. Data is presented as the mean ± SEM (n=3). Normalized AUC x Log [6-OHDA] (M) Figure 7. Human ipsc-derived neurones in co-culture. icell Neurons (CDI) co-cultured with either human ipsc-derived astrocytes (A, C, E, icell Astrocytes, CDI) or rat primary astrocytes (, D, F, CellPlayer NeuroPrime rastrocytes). Phase contrast images and the corresponding red fluorescent images are shown. The time-courses of neurite development are similar when either ipsc-derived (E) or rat primary astrocytes (F) are used in co-culture (mean + SEM, N = 4-6). Utility of NeuroLight Red with ipsc-derived neuronal coculture models Advancements in stem cell research have lead to an increase in the availability of human ipsc-derived cell types, which now offer the potential to develop phenotypic disease models In an effort to develop a translational model of PD, we created a co-culture system of human ipsc-derived dopaminergic neurons (icell DopaNeurons) with rat primary astrocytes (Figure 8). Following a period of neurite development (4 days), the dopaminergic-specific neurotoxin 6-OHDA was applied to effect neuronal damage. A time- and concentrationdependent neurite disruption was observed, yielding an IC 5 value for 6-OHDA of 34 µm. At the maximum tested concentration of 6-OHDA (5 µm), neurites were abolished (95% inhibition), measuring 2.8 ±.3 mm/mm 2 in the presence of 6-OHDA compared to 58 ± 3 mm/mm 2 in the vehicle controls. This model system may provide a quantitative phenotypic assay for agents designed to reverse or minimize the neurotoxic effects of 6-OHDA and aid in the development of improved PD therapeutics.

6 6 CellPlayer 96-Well Kinetic NeuroTrack Co-culture Assay Conclusions References The IncuCyte ZOOM live-cell imaging platform, combined with NeuroTrack software and the NeuroLight Red labeling reagent, provides a means to image neurons in co-culture with astrocytes and quantitate changes in neurite length of living cultures from days to weeks. This approach provides a sensitive method to detect pharmacological manipulations that alter neurite dynamics, including processes such as elongation and retraction. NeuroLight Red can be used with multiple neuronal types providing flexibility in assay design. The NeuroPrime Cell Kit, in conjunction with the NeuroTrack software module, also provides a ready to use and optimized solution for measuring kinetic changes in neurite length using primary neurons. The imaging strategy allows all quantitative data to be validated by direct comparison with morphological changes in the images, allowing a uniquely thorough analysis in a single assay. 1. The image analysis software is flexible for quantification of many cells types, from large immortalized cell lines to primary neurons and ipscs. An intuitive user interface allows rapid assay optimization and automated quantification of neurite dynamics. 2. All data and time points can be verified by inspecting individual images and/or time-lapse movies. Observation of cell morphology provides additional validation and insight into the biological effect of treatment groups. 3. Kinetic measurements of neurite length, branch points, and cell body clusters capture a complete description of a highly dynamic process that cannot be provided by single time point data alone. Arbitrarily defined endpoints are not required. 4. Low well-to-well variability and minimal user effort make the assay amenable to medium throughput screening in a 96-well format. Choi DW (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: Conforti L, Adalbert R and Coleman MP (27) Neuronal death: where does the end begin? Trends Neurosci 3: Huettner JE and ean P (1988) lock of N-methyl-Daspartate-activated current by the anticonvulsant MK- 81: selective binding to open channels. Proc Natl Acad Sci U S A 85: Li LH, Qin HZ, Wang JL, Wang J, Wang XL and Gao GD (29) Axonal degeneration of nigra-striatum dopaminergic neurons induced by 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine in mice. J Int Med Res 37: Mattson MP (28) Glutamate and neurotrophic factors in neuronal plasticity and disease. Ann N Y Acad Sci 1144: Skibinski G, Nakamura K, Cookson MR and Finkbeiner S (214) Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies. J Neurosci 34: Winner, Jappelli R, Maji SK, Desplats PA, oyer L, Aigner S, Hetzer C, Loher T, Vilar M, Campioni S, Tzitzilonis C, Soragni A, Jessberger S, Mira H, Consiglio A, Pham E, Masliah E, Gage FH and Riek R (211) In vivo demonstration that alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A 18: Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN and Iversen LL (1986) The anticonvulsant MK-81 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci U S A 83:714-8.

7 CellPlayer 96-Well Kinetic NeuroTrack Assay 7 NOTES A