Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

Abstract

Accurate quantification of synaptic changes is essential for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and synaptic toxicity. Here we demonstrate a robust high-content imaging method for the assessment of synaptic changes and apply the method to brain homogenates from an Alzheimer's disease mouse model. Our method uses serial imaging of endogenous fluorescent labeled presynaptic VAMP2 and postsynaptic PSD95 in long-term cultured live primary neurons in 96 well microplates, and uses automatic image analysis to quantify the number of colocalized mature synaptic puncta for the assessment of synaptic changes in live neurons. As a control, we demonstrated that our synaptic puncta assay is at least 10-fold more sensitive to the toxic effects of glutamate than the Mtt assay. Using our assay, we have compared synaptotoxic activities in size-exclusion chromatography fractioned protein samples from 3xTg-AD mouse model brain homogenates. Multiple synaptotoxic activities were found in high and low molecular weight fractions. Amyloid-beta immunodepletion alleviated some but not all of the synaptotoxic activities. Although the biochemical entities responsible for the synaptotoxic activities have yet to be determined, these proof-of-concept results demonstrate that this novel assay may have many potential mechanistic and therapeutic applications. Synapses are asymmetric intercellular junctions that permit neurons to transmit electrical or chemical signals to other neurons. Dysregulated synaptic function, abnormal synaptic plasticity and synaptic loss are involved in many neurodevelopmental and neurodegenerative disorders, in particular Alzheimer's disease (AD) 1-3. As the most common dementing disorder in the elderly, AD is tightly correlated with synaptic loss in vulnerable brain regions, which has led to the hypothesis that loss of synaptic terminals is a key event in early cognitive decline 4. Accurate quantification of mammalian central nervous system synaptic changes is important for understanding the molecular mechanisms of synaptogenesis, synaptic plasticity, and developmental or pathological synapse elimination. As many neurodegenerative conditions are known to affect synapses, quantification of synapses is also critical for identifying synaptotoxic factors and for screening of potential therapeutic agents. However, few efficient methods are available for this purpose. Electrophysiology, electron microscopy, and most light microscopy approaches are costly, time-consuming, and labor-intensive. Unbiased, high throughput screening for synaptotoxic substances in brain disorders such as AD would provide a better understanding of the disease process and an opportunity to identifying new therapeutic targets and drug candidates. High content screening (HCS) combines the efficiency of high-throughput, multi-well plate-based techniques with quantitative image analysis at subcellular resolution 5-7. Recent advances in HCS have made high-throughput analysis of synaptic changes in live neurons po...

Depression patient-derived cortical neurons reveal potential biomarkers for antidepressant response

“…Together, these synaptic changes could be used as a biomarker for bupropion effects in vitro. It is noteworthy that microscopy-based high content screening has been shown to be feasible to detect synaptic changes 47 , further highlighting the potential use of our results.…”

Unbiased high-content screening reveals Aβ- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer’s patients and high-pathology controls

“…Interestingly, synaptotoxic activities were identified in all samples including CDR0 normal controls (Figs 1I and 4 ). Human brain SEC fractions that showed synaptotoxic activities were distributed from 17 to 20 mL of the total SEC eluent, which indicates larger molecular weight components than the synaptotoxic fractions identified in 3xTg-AD mouse brain lysates assessed under identical conditions (fractions 20–22) [ 23 ]. Furthermore, the fractions with synaptic toxicity were slightly larger than those containing Aβ monomer (fractions 18–21, Fig 3B ), and smaller than the peak of the tau distribution (fractions 16–17, Fig 3C ).…”

“…Plates were then washed with sterile distilled water three times and dried for at least 30 min before use. Hippocampal neurons were collected and cultured as described [ 23 ]. To minimize evaporation and edge effects on the microplate during imaging, the interwell region of the culture plate was filled with sterile water.…”

“…Evaluation of the nature of neuronal and synaptic changes in cognitively normal individuals with AD pathology and in AD cases may provide an advantage for identification of synaptotoxic substances and for understanding of the progression AD. We recently developed a robust high-content imaging method for assessing synaptic changes in a 96 well plate format [ 23 ]. Our method uses serial imaging of endogenous labeled presynaptic VAMP2-mRFP [ 24 ] and postsynaptic PSD95-mVenus [ 25 ] protein in long-term cultured murine primary neurons to quantitate the number of synaptic puncta for the assessment of synaptic changes ( Fig 1 ).…”

“…We previously showed that multiple synaptotoxic activities can be detected in size-exclusion chromatography (SEC) fractioned brain homogenates from 3xTg-AD mice [ 26 ]. Interestingly, both Aβ-related and apparently Aβ-independent synaptotoxic activities have been identified [ 23 ]. However, the synaptotoxicities in human brain homogenates have not been assessed in this fashion.…”