Angiotensin-converting enzyme 2 (ACE2) has been identified as a critical receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has led to extensive speculation on the role of ACE2 in disease severity, and in particular, whether variation in its expression can explain higher mortality in older individuals. We examine this question in mouse lung and show that 24-month old mice have significantly reduced ACE2 mRNA expression relative to 3-month old mice. The differences appear to be localized to ciliated cells

Booeshaghi, A. Sina

Pachter, Lior

English

Caltech Authors - Main

 Decrease in ​ACE2​ mRNA expression in aged mouse lung 
 
A. Sina Booeshaghi ​1​ and Lior Pachter​2,3,* 
 
1. Department of Mechanical Engineering, California Institute of Technology, Pasadena, CA,  
2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 
3. Department of Computing and Mathematical Sciences, California Institute of Technology, Pasadena, CA 
*Address correspondence to lpachter@caltech.edu 
 
Angiotensin-converting enzyme 2 (​ACE2​) has been identified as a critical receptor for severe             
acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has led to extensive speculation            
on the role of ​ACE2 in disease severity, and in particular, whether variation in its expression can                 
explain higher mortality in older individuals. We examine this question in mouse lung and show               
that 24-month old mice have significantly reduced ​ACE2 mRNA expression relative to 3-month             
old mice. The differences appear to be localized to ciliated cells. 
 
Introduction 
The severity of illness resulting from infection with        
SARS-CoV-2 virus​1 is strongly correlated with age ​2​.       
There has been much speculation about the       
mechanisms underlying this strong association, with      
one hypothesis being that age-related changes in       
ACE2​, a crucial receptor used by SARS-CoV-2 to        
enter cells​3​, affect disease susceptibility​4​.     
Nevertheless, there has been little data on the age         
dependence of ​ACE2 expression in humans, with the        
exception of GTEx data, which is limited in the age          
range to adults​5​. 
 
To better assess the relationship between age and        
ACE2 expression, we examined single-cell RNA-seq      
data from eight 3-month old and seven 24-month old         
mouse lungs​6 in order to identify cell-type specific        
age-related differences, if any, in ​ACE2 mRNA       
expression. Single-cell RNA-seq can determine     
whether bulk shifts in gene expression are due to         
changes in cell-type abundances, or changes in gene        
expression within specific cell types​7​. We find that        
both cell-type abundance shifts, as well as ​ACE2        
mRNA changes in specific cell types, contribute to a         
decrease in ​ACE2 ​mRNA with age. We corroborate        
our findings with bulk RNA-seq analysis and ​ACE2        
protein measurement in rat lungs​8​. 
 
Results 
To examine mRNA abundance of ​ACE2 in young and         
old mice lungs, we began by uniformly processing ​9,10        
all RNA-seq from GSE12487 ​6 (see Methods),      
followed by computation of common coordinates for       
the 3-month and 24-month old mice single-cell       
RNA-seq using scVI​11 (Figure 1a, Methods).      
Consistent with previous findings​12​, we find that       
ACE2 is not prevalent in the lung, with the exception          
of Goblet/Club and ciliated cells (Supp. Fig. 1). In         
cells where ​ACE2 ​mRNA molecules are identified,       
the average copy number is 1.19. We, therefore,        
measured the expression level of ​ACE2 ​by the        
proportion of cells with at least one copy (Methods). 
 
In a comparison of the 3-month old and 24-month old          
mice, we found significantly fewer cells with ​ACE2        
expression in the 24-month old mice (2.1 fold        
reduction, p = 0.0375, Figure 1b). We corroborated        
this result with an analysis of three bulk RNA-seq         
samples from 3-month old mice and three bulk        
RNA-seq samples from 22-month old mice (1.5 fold        
reduction, p = 0.0163, Supp. Fig. 2). This reduction in          
ACE2 ​mRNA expression is also seen in an analysis         
of FACS sorted alveolar macrophage bulk RNA-seq       
samples (2.7 fold reduction, p = 0.3076, Supp. Fig 3).          
These results are consistent with a previously       
 
.CC-BY 4.0 International licenseauthor/funder. It is made available under a
The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.04.02.021451doi: bioRxiv preprint 
 
Figure 1: ​a) ​ACE2​ mRNA expression in a tSNE representation of scVI coordinates for 3-month and 24-month old mice. b) 
Proportion of cells displaying ​ACE2​ in 3-month old vs. 24-month old mice. c) ​ACE2​ mRNA expression by cell type in 
young vs. old mice (inset: ratio of Goblet/Club cells to ciliated cells in 3-month old vs. 24-month old mice). 
 
observed dramatic reduction of ​ACE2 ​protein      
between 3-month old and 24-month old rat lungs​8​. 
 
The decrease in ​ACE2 mRNA expression with age in         
lungs is likely due to two underlying phenomena: a         
reduction of ​ACE2 mRNA in ciliated cells, and a shift          
in ciliated cell abundance with age (Figure 1c, Supp.         
Fig 4). The latter result is statistically significant and         
corroborated in an analysis of FACS sorted cells​6​.  
 
SARS-CoV-2 uses the serine protease ​TMPRSS2 for       
Spike protein priming, so we repeated our analyses        
for ​TMPRSS2​. We found no significant changes in        
mRNA expression between 3-month and 24-month      
old mice (Supp. Figs. 5-7).  
 
Discussion 
Our results show a decrease in ​ACE2 mRNA        
expression between young and old mice and       
contradict recent claims by others​13​. To understand       
the discrepancies in results, we examined differences       
in data or methodology that could lead to        
contradictory results (Supp. Table 1). We conclude       
that there is strong evidence in support of a decrease          
in ​ACE2 ​mRNA between young and old mice, and         
that other currently available RNA-seq datasets do       
not contradict this finding. We note that results in         
rodents may not necessarily translate to humans. 
 
Methods 
All methods and code to generate the results in this          
manuscript can be found in the python notebooks at         
https://github.com/pachterlab/BP_2020 ​. 
 
Data availability 
Raw FASTQ files can be obtained from ​GSE124872 ​. 
 
References 
1. Zhou, P. ​et al. ​ A pneumonia outbreak associated with a new coronavirus of probable bat 
origin. ​Nature​ ​579, ​ 270–273 (2020). 
2. Yang, X. ​et al. ​ Clinical course and outcomes of critically ill patients with SARS-CoV-2 
pneumonia in Wuhan, China: a single-centered, retrospective, observational study. ​Lancet 
Respir. Med.​ (2020). doi:10.1016/S2213-2600(20)30079-5 
3. Yan, R. ​et al. ​ Structural basis for the recognition of the SARS-CoV-2 by full-length human 
ACE2. ​Science ​ (2020). doi:10.1126/science.abb2762 
4. Schatzmann Peron, J. P. & Nakaya, H. Susceptibility of the Elderly to SARS-CoV-2 Infection: 
ACE-2 Overexpression, Shedding and Antibody-dependent Enhancement (ADE). (2020). 
doi:10.20944/preprints202003.0400.v1 
5. Chen, J. ​et al. ​ Individual Variation of the SARS-CoV2 Receptor ACE2 Gene Expression and 
Regulation. (2020). 
6. Angelidis, I. ​et al.​ An atlas of the aging lung mapped by single cell transcriptomics and deep 
tissue proteomics. ​Nat. Commun.​ ​10, ​ 963 (2019). 
7. Trapnell, C. Defining cell types and states with single-cell genomics. ​Genome Res.​ ​25, 
1491–1498 (2015). 
8. Xie, X., Chen, J., Wang, X., Zhang, F. & Liu, Y. Age- and gender-related difference of ACE2 
expression in rat lung. ​Life Sci.​ ​78, ​ 2166–2171 (2006). 
9. Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq 
quantification. ​Nat. Biotechnol.​ ​34, ​ 525–527 (2016). 
10. Melsted, P., Booeshaghi, A.S. ​et al. ​ Modular and efficient pre-processing of single-cell 
RNA-seq. ​BioRxiv​ (2019). doi:10.1101/673285 
11. Lopez, R., Regier, J., Cole, M. B., Jordan, M. I. & Yosef, N. Deep generative modeling for 
single-cell transcriptomics. ​Nat. Methods​ ​15, ​ 1053–1058 (2018). 
12. Sungnak, W., Huang, N., Bécavin, C., Berg, M. & Network, H. L. B. SARS-CoV-2 Entry 
Genes Are Most Highly Expressed in Nasal Goblet and Ciliated Cells within Human Airways. 
arXiv​ (2020). 
13. Smith, J. C. & Sheltzer, J. M. Cigarette smoke triggers the expansion of a subpopulation of 
respiratory epithelial cells that express the SARS-CoV-2 receptor ACE2. ​bioRxiv​ (2020). 
.CC-BY 4.0 International licenseauthor/funder. It is made available under a
The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.04.02.021451doi: bioRxiv preprint 


Decrease in ACE2 mRNA expression in aged mouse lung

https://authors.library.caltech.edu/102345/1/2020.04.02.021451v1.full.pdf

Decrease in ACE2 mRNA expression in aged mouse lung

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main