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Aging and disease    2019, Vol. 10 Issue (6) : 1146-1158     DOI: 10.14336/AD.2019.0225
Orginal Article |
AmpliSeq Transcriptome of Laser Captured Neurons from Alzheimer Brain: Comparison of Single Cell Versus Neuron Pools
Wenjun Deng1,2,*, Changhong Xing1,3,*, Rob David4, Diego Mastroeni5, MingMing Ning1,2, Eng H Lo1, Paul D Coleman5,*
1Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02192, USA
2Clinical Proteomics Research Center, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
4Thermo-Fisher Scientific, Salem, MA 02114, USA
5ASU-Banner Neurodegenerative Disease Research Center, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA
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Abstract  

Alzheimer’s disease (AD) is the most common cause of dementia in older adults. However, the pathogenesis of AD remains to be fully understood and clinically effective treatments are lacking. Recent advances in single cell RNA sequencing offers an opportunity to characterize the heterogeneity of cell response and explore the molecular mechanism of complex diseases at a single cell level. Here, we present the application of the Ion AmpliSeq transcriptome approach to profile gene expression in single laser captured neurons as well as pooled 10 and 100 neurons from hippocampal CA1 of AD brains versus matching normal aged brains. Our results demonstrated the high sensitivity and high genome coverage of the AmpliSeq transcriptome in single cell sequencing. In addition to capturing the known changes related to AD, our data confirmed the diversity of neuronal profiles in AD brain, which allow the potential identification of single cell response that might be hidden in population analyses. Notably, we also revealed the extensive inhibition of olfactory signaling and confirmed the reduction of neurotransmitter receptors in AD hippocampus. We conclude that although single neuron data show more variance than data from 10 or 100 pooled neurons, single neuron data can be informative. These findings support the utility of the Ion AmpliSeq method for obtaining and analyzing gene expression data from single defined laser captured neurons.

Keywords AmpliSeq transcriptome      single neuron      Alzheimer's disease     
Corresponding Authors: Deng Wenjun,Xing Changhong,Coleman Paul D   
Just Accepted Date: 12 April 2019   Issue Date: 16 November 2019
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Deng Wenjun
Xing Changhong
David Rob
Mastroeni Diego
Ning MingMing
Lo Eng H
Coleman Paul D
Cite this article:   
Deng Wenjun,Xing Changhong,David Rob, et al. AmpliSeq Transcriptome of Laser Captured Neurons from Alzheimer Brain: Comparison of Single Cell Versus Neuron Pools[J]. Aging and disease, 2019, 10(6): 1146-1158.
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http://www.aginganddisease.org/EN/10.14336/AD.2019.0225     OR     http://www.aginganddisease.org/EN/Y2019/V10/I6/1146
SamplesMapped readsOn targetTargets detectedGenes Identified
Single neuron_15,416,54290.65%61.51%15,094
Single neuron_25,871,99581.56%64.82%15,586
Single neuron_35,748,51267.48%64.16%15,582
10 neurons_16,153,10983.52%66.98%16,375
10 neurons_25,793,32683.08%65.61%16,075
10 neurons_35,596,52884.35%65.69%16,662
100 neurons4,239,54489.57%64.90%16,764
Table 1  Summary of AD neuron transcriptome.
Figure 1.  Evaluation of AmpliSeq transcriptome sequencing results. (A) Comparison of gene identifications between Ion AmpliSeq technology and other RNA sequencing platform. (B) The distribution of gene abundance in single neuron and pooled 10 and 100 neurons (left) as well as in single neurons with different loading amount (right). (C) Pearson correlation of gene expression between technical replicates. (D) Comparison of gene expression quantified by AmpliSeq with the hippocampus neuron transcriptome measured in other species using different technologies.
Figure 2.  Gene expression diversity in different neuron sets. Pearson correlation of gene expression between replicate experiments (A) and across different neuron sets (B). (C) Principal component analysis of gene expression profiles in different neuron sets. (D) Standard deviation of gene expression within replicates binned according to gene expression levels (mean±95% CI).
PlatformNeuron originNeuron setsReplicatesIdentified genes
MinimumMaximumAverageSD
ThermoFisher Ion AmpliSeq transcriptomeHuman
hippocampus CA1
Single cell315,09415,58615,421283
10 cells316,07516,66216,371294
100 cells1N/AN/A16,764N/A

Illumina MiSeq
Krishnaswami et al. 2016
Human brainSingle cell63,3856,2674,5671,209

Illumina HiSeq 2000
Nichterwitz et al. 2016
Human
motor neuron
Single cell48,4559,2088,795310
5 cells98,68712,23710,7721,040
10 cells510,60512,50011,482921

Illumina HiSeq 2500
Lacar et al. 2016
Mouse
dentate granule cell
Single cell384,5538,7696,8431,135
Table 2  Comparison with other single neuron sequencing studies.
Figure 3.  The expression diversity of neuronal markers in different neuron sets. (A) The relative expression of neuron markers across the different neuron sets. (B) Examples of neuron marker expression in single neurons and pooled 10 and 100 neurons.
Figure 4.  AmpliSeq neuron transcriptome in AD versus control. (A) Principal component analysis of gene expression between AD and control neurons. (B) The expression levels of well-characterized AD genes in AD and control neurons. (C) Correlation of gene expression changes across different neuron sets. (D) The Venn diagram of the genes with expression change in AD brain across different neuron sets.
Figure 5.  AD-related functional alterations in different neuron sets. (A) Biological functions enriched with the genes with >2-fold expression changes in AD brain. (B) The heatmap of genes involved in apoptosis. (C) The expression levels of well-characterized pro-apoptotic genes in AD and control neurons.
Figure 6.  Olfactory transduction was significantly inhibited in AD neuron. (A) The heatmap of genes involved in olfactory signal transduction. (B) The expression distribution of olfactory receptor family in the genome of control and AD neuron. (C) The heatmap of significantly changed olfactory receptors. (D) The expression levels of the olfactory receptors with the highest abundance as well as β-arrestin 2 in AD and control neurons.
Figure 7.  Neurotransmitter receptors were reduced in AD neurons. (A) The functional classification of the genes involved in G-protein coupled receptor signaling pathway. (B) The expression levels of the receptors of dopamine, GABA and 5-hydroxytryptamine neurotransmission system.
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