Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

<div><p>Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca²⁺ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.</p></div

Increased extracellular Ca²⁺ concentration sensitizes CGNs to excitotoxic insult and induces cell death mainly through apoptosis.

<p>CGNs were seeded at 50,000 cells/well and cultured <i>in vitro</i> for 7/8 days. CGNs were treated with different glutamate (Glut.) and Ca²⁺ concentrations as indicated for 10 min. A) The population (Popul.) of CGNs (%), for each glutamate and Ca²⁺ concentration, classified as viable (blue traces), apoptotic (green traces) or necrotic (red traces). Traces shown are median ± inter-quartile regions. Neurons exposed to high glutamate and high Ca²⁺ were more sensitive to cell death. B) A heatmap of median cell viability 24 h following glutamate exposure illustrates lower viability in CGNs exposed to increasing glutamate and extracellular Ca²⁺ concentrations. C) Populations of viable cells 24 h following glutamate excitation. Neurons exposed to 2.0 mM Ca²⁺ (white boxes) were more vulnerable to glutamate excitation. Boxplots show the median ± inter-quartile regions (n = 10 wells for each treatment, from 5 independent experiments). D) Populations of viable, apoptotic and necrotic neurons 24 h following exposure to 100μM or 300μM glutamate at 1.5 and 2.0 mM extracellular Ca²⁺. Neurons exposed to 300 μM glutamate and 2.0 mM Ca²⁺ show increased necrotic cell death. E) Heat map illustrating prolonged glutamate exposure induced cell death at 24 h post glutamate excitation. F) Distribution of viable apoptotic and necrotic neurons in response to prolonged glutamate excitation (100 μM /10 μM glycine) at 1h and 24 h post excitation. Quantification from 4 wells from 2 independent experiments. Boxplots show the median ± inter-quartile regions. Boxplot shows increase in apoptotic population in response to prolonged glutamate excitation. Total 8 wells from 4 independent experiments.</p

CGNs seeded either at low or high seeding densities undergo increased necrosis in response to glutamate excitation.

<p>Cerebellar granule neurons (CGNs) seeded at 25,000, 50,000 or 100,000 cells/well were cultured for 7/8 DIV and treated with 10, 30 or 100 μM glutamate (Glut; with 10 μM glycine) for 10 min before media was replaced with high Mg²⁺ (1.2 mM) containing buffer to block glutamate receptors. For 24 h following glutamate exposure, 9 fields of view per well (550–650 cells/field of view) were imaged at 1 hr intervals, and neurons were classified as viable, apoptotic or necrotic based on their nuclear morphology (Hoechst) and plasma membrane integrity (PI). A) Representative transmitted light images of CGNs seeded at 25,000, 50,000 or 100,000 cells/well in 96-well plates and cultured <i>in vitro</i> for 7 days. Scale bar 40 μm. B) The population of cells (%), for each density and glutamate treatment, classified as viable (blue traces), apoptotic (green traces) or necrotic (red traces). Traces are median ± inter-quartile regions of all wells exposed to the same treatment. C) Quantification of viable, apoptotic and necrotic populations 24 h following glutamate exposure for each of the seeding densities and glutamate concentrations. Boxplots show the median ± inter-quartile regions (n = 8 wells for each treatment, from 4 independent experiments). Neurons seeded at 50,000 cells/well had lower well-to-well variability than neurons seeded at 25,000 or 100,000 cells/well, and underwent less necrotic cell death in response to glutamate excitotoxicity. Neurons seeded at 50,000 cells/well and treated with 100 μM glutamate underwent significantly increased apoptosis compared to neurons treated with 10 μM glutamate (*p = 0.014).</p

Classification and validation of viable, necrotic and apoptotic population in CGNs.

<p>A) The high content screening workflow detected mostly viable cells (blue traces) when neurons were treated with control media. B) Treating wells with 0.01% Triton-x-100 for 10 min to induce necrosis prior to imaging resulted in most cells being classified as necrotic (red traces). C) Treating wells with the apoptosis inducing staurosporine (STS) at 30 nM concentration for 24 h prior to the start of image acquisition, resulted in cells primarily being classified as apoptotic (green traces). Each graph represents 9 fields of view (550–650 cells / field of view), taken from each well for 24 h with 1 h time interval between each scan. Graphs are representative of 2 independent experiments. Two individual wells are shown to demonstrate the consistency of classification across wells. D) Representative Hoechst, PI and brightfield images at 0 h and 24 h following 100 μM glutamate treatment for 10 min. Quantification of viable, apoptotic and necrotic populations in these wells (bar charts) demonstrated that neuronal cell death 24 h after transient glutamate exposure was primarily apoptotic.</p

Decreased glucose availability increased sensitivity of neurons to glutamate excitation.

<p>Neurons seeded at 100,000 cells/well were cultured for 7 DIV in 4 mM, 6 mM or 15 mM glucose and treated with 10, 30 or 100 μM glutamate (Glut.), as indicated, for 10 minutes. A) Representative brightfield images showing that neurons cultured in lower glucose did not affect basal cell viability. B) The population of neurons (%) classified as viable (blue traces), apoptotic (green traces) and necrotic (red traces). Median traces are shown from two wells for each treatment. Neurons cultured in low glucose conditions are more sensitive to glutamate excitation. C) The population (%) of viable, apoptotic and necrotic neurons in each well 24 h following glutamate excitation. Neurons cultured in 4 mM glucose underwent increased necrosis. D) A heat map showing the median population of viable cells 24 h following glutamate exposure.</p

Pharmacological inhibition of cell death pathways protects neurons against glutamate-induced cell death.

<p>Neurons were seeded at 50,000 cells/well and cultured <i>in vitro</i> for 7/8 days. Neurons were pre-treated for 1h prior to glutamate excitation with calpeptin (20 nM), rapamycin (250 nM), DPQ (100 μM), Compound C (10 μM) or SP600125 (5 μM). Following glutamate exposure (Glut.; 100 μM for 10 or 30 min,), media was replaced with pre-conditioned media alone (in the case of Compound C and SP600125) or pre-conditioned media containing the drugs, and images were acquired for 24 h. A) Populations (Popul.) of neurons classified as viable (blue traces), apoptotic (green traces) and necrotic (red traces). Traces show the median ± inter-quartile regions (n = 4 wells from 2 independent experiments). Traces show that blocking key cell death signalling pathways protected neurons against excitotoxicity. B) A heat map of the median population of viable cells 24 h following glutamate excitation (100 μM) illustrates the protective effect of these drugs against glutamate-induced cell death. C) Boxplots showing % of viable, apoptotic and necrotic neurons 24 h following 100 μM glutamate excitation for 10 min. Boxplots show the median ± inter-quartile regions. Pre-treatment with Calpeptin, DPQ and SP 600125 significantly increased viability compared to no pre-treatment and this increased viability was primarily due to a decrease in apoptosis (*p = 0.0475 for all marked treatments compared to no pre-treatment).</p

High content screening (HCS) workflow: Background correction, segmentation and classification of Hoechst and PI stained neurons in real time HCS experiments.

<p>A) Representative images of mouse cerebellar granular neurons cultured in 96 well plates and stained with (i) Hoechst (100 ng/ml for 1 h) and (ii) PI (250 ng/ml). Variations in background signal due to illumination fluctuation were corrected using CellProfiler. Scale bar: 40 μm. B) Time-lapse images obtained from HCS experiments were processed using CellProfiler and MATLAB. i) Flow chart showing steps involved in analysis of stained neurons following image acquisition on the HCS system—nuclei segmentation and measuring, tracking objects over time, and classification and validation of time-lapse images from HCS. ii) Representative cells were selected for classification by a human expert—nuclei characteristics were extracted and used as a training set for unsupervised classification by binary decision tree. Nuclei were categorised as: Class 1 –when the Hoechst area was large and uniform with even staining; or Class 2 –apoptotic cells showing condensed nuclei and intense Hoechst staining. Based on PI fluorescence intensity, objects in Class 1 were further distinguished as viable (low PI) and necrotic (high PI) neurons. iii) Changes in classification of each cell over time were tested and validated. Green and red arrows indicate valid and invalid state transitions, respectively. C) Neurons were segmented using a combination of Otsu thresholding and watershed algorithm, implemented in CellProfiler. (i) Representative Hoechst image of a field of view acquired by HCS, showing masks for Hoechst stained nuclei (green outlines). Scale bar: 40 μm. (ii) Representative Hoechst image (zoomed in white box in i), object masks and segmented object areas from CellProfiler. Green masks represent objects that were measured for further analysis while objects showing red outlines were discarded due to size restrictions, mostly caused by under-segmentation of clumped cells. Scale bar: 10 μm. (iii) Time lapse image series of Hoechst stained neurons showing variation in segmentation. Annotation (white arrow) 1 & 2 show identification and segmentation of previously unsegmented neurons. Annotation 3 shows inaccurate object segmentation which had been correctly segmented in previous timepoints.</p

List of compounds used in this study.

<p>List of compounds used in this study.</p