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Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons.

ABSTRACT: Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

SUBMITTER: Snowden SG

PROVIDER: S-EPMC7644967 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of <i>SNCA-A53T</i> Human Dopamine Neurons.

Snowden Stuart G SG Fernandes Hugo J R HJR Kent Josh J Foskolou Stefanie S Tate Peri P Field Sarah F SF Metzakopian Emmanouil E Koulman Albert A

iScience 20201021 11

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolut ...[more]

PMID: 33196026

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Project description:Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

Dataset Information

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons.

Publications

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of <i>SNCA-A53T</i> Human Dopamine Neurons.

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