†These authors contributed equally.
Academic Editors: Rafael Franco and Jolanta Dorszewska
Background: Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. Methods: In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. Results: Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. Conclusions: Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.
Nogo is a myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein. It belongs to the reticulon (RTN) protein family and has three different subtypes: Nogo-A, Nogo-B and Nogo-C. These molecules are thought to inhibit neuronal regeneration in the central nervous system (CNS) through binding to Nogo-66 receptor (NgR1) [1, 2, 3, 4]. NgR1 is a leucine-rich repetitive protein encoded by the RTN4R gene and is mainly expressed in the body of nerve cells and muscle in vivo [5, 6, 7, 8]. It has a high affinity for Nogo-66, which is the main inhibitory region of Nogo-A [6, 9, 10]. Nogo-A is mostly expressed by myelinated oligodendrocytes in the CNS . Nogo-66 binding to NgR1 plays a key role in inhibiting neurite growth and in limiting synaptic plasticity [6, 12, 13]. Other studies have suggested that Nogo and its receptor are associated not only with neurite regeneration but also with neurodegenerative disorders, such as Alzheimer’s disease (AD) [10, 14, 15]. A study by Dermody  first identified NgR1 as a neural receptor for Mammalian Reovirus (MRV) that mediated infection by this virus.
So far, few animals that have proved suitable as models for CNS virus infection. The main MRV animal model currently in use is the mouse. Although this model provides a pathological basis for neurotropic virus nerve transmission, the molecular mechanism of nerve trafficking by the virus remain unclear. The Chinese tree shrew (Tupaia belangeri chinensis) is a new and valuable experimental animal model due to its low maintenance cost and short breeding cycle. It has a high brain-to-body mass ratio  and is used to study human conditions and diseases such as brain development and aging [17, 18, 19], depression , social stress , AD [16, 22, 23, 24], Parkinson’s disease (PD) , and MRV infection [26, 27]. We previously isolated two new MRV strains from Chinese tree shrews and found these animals were susceptible to MRV infection . The Chinese tree shrew is therefore a good animal model to study viral infection of CNS system. However, the mechanism of MRV infection is still unknown, while the molecular characterization and function of NgR1, one of the receptors for MRV infection, remains unclear.
To date, NgR1 from the human, chick, mouse and fish have been cloned [8, 28]. However, little is known about the structure and function of tree shrew NgR1. In the present study, we determined the sequence of tree shrew NgR1 by RACE technology, described its biochemical characterization, predicted its spatial structure, and examined its expression pattern in different tissues. We also studied the function of NgR1 in tree shrew primary neuronal cell (tNC) in response to MRV1/TS/2011 infection. Our results indicate that NgR1 has a closer relationship to human NgR1 than to mouse NgR1, and that NgR1 can mediate infection by MRV1/TS/2011.
Chinese tree shrews (1–3 days old newborns and 1–2 years old adults) were raised at the Center for Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China. After euthanasia with an overdose of pentobarbital sodium, 26 different tissues (brain, tongue, esophagus, heart, lung, liver, gallbladder, stomach, spleen, duodenum, jejunum, ileum, caecum, colon, rectum, kidney, bladder, testis, parietal lobe, cerebellum, temporal lobe, hippocampus, frontal lobe, optic chiasma, occipital lobe and blood) were dissected immediately, quickly frozen in liquid nitrogen and stored at –80 °C. This research project was approved by the institutional ethics committee and all procedures were conducted in accordance with ethical standards and practices.
Primers were designed according to the predicted Tupaia chinensis
reticulon 4-receptor (RTN4R) registered in GenBank (Supplementary Table
1). The detailed protocol can be found in our previous reports . Briefly,
RNA was extracted from brain tissue and the 5
The MEGA 7.0 program (Mega Limited, Auckland, New Zealand) was used to analyze the homology of NgR1 in tree shrew, human and several other common experimental animals (8 species). The NgR1 phylogenetic tree was constructed using MEGA 7.0 (Mega Limited, Auckland, New Zealand), with Danio rerio used as the outgroup. The accuracies and statistical tests tree branch position were tested using the bootstrap method with 1000 replications.
The predicted three-dimensional structure of NgR1 was constructed using SWISS-MODEL integrated with the visualization program PyMOL2.2.0 (DeLano Scientific, San Carlos, CA, USA). The full-length protein sequence was determined using MEGA7.0 (Mega Limited, Auckland, New Zealand). Tree shrew NgR1 protein sequence was imported into the SWISS-MODEL online analysis platform (https://swissmodel.expasy.org/) to generate a protein model PDB format file, with PyMOL used to visualize the 3D model. Search and download of the human NgR1 protein structure (Q9BZR6) template was performed using UniProt (https://www.uniprot.org/). The three-dimensional structures for tree shrew and human NgR1 were superimposed using PyMOL/align.
Total RNA was prepared using MRC RNAzol (MRC, Cincinnati, OH, USA) according to the manufacturer’s instructions. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed with gene specific primers (Supplementary Table 2) as reported previously  and using tsGAPDH as an internal control. Relative NgR1 gene expression was calculated using the double-standard curve method.
The preparation and culture of tree shrew primary neuronal cells (tNC) was
described in our previous study . Briefly, brain tissue was washed twice with
pre-cooled PBS, cut into 1 mm blocks using micro-scissors and then digested with
0.25% trypsin (Gibco, Carlsbad, CA, USA) for 20 min. The digestion was stopped with DMEM /High
Glucose (Hyclone, Logan, UT, USA) +5% FBS (Hyclone, Logan, UT, USA), and the cells centrifuged at 2000
g for 10 min. tNCs were resuspended and cultured at a density of 5
Tree shrew-derived Mammalian Reovirus (MRV1/TS/2011, 10
The primary neuronal cells derived from tree shrew (tNC) were divided into 8
groups according to the treatment used: in group A, only PBS was added; group B,
Neuraminidase (NA); group C, Phosphatidylinositol specific phospholipase C
(PI-PLC, 50 mU/mL); group D, PI-PLC (100 mU/mL) and NA (50 mU/mL); group E,
Anti-Nogo Receptor antibody (ab32890, 50
Twenty-four hours post infection, tNC cells were fixed with 1 mL of 4%
paraformaldehyde for 20 minutes and the antiviral antigen
One-way ANOVA was used to analyze for differences between groups. A significant
difference was scored as **** p
The NgR1 gene sequences were comprised of 3128 bp cDNA and coded for 731 amino acids (Supplementary Fig. 1). Multiple sequence alignment (MSA) revealed that NgR1 was more homologous to human (90.34%) and Macaca fascicularis (89.91%) or Macaca mulatta (89.91%) than it was to mouse (89.06%) or rat (88.84%) NgR1 (Supplementary Fig. 2). Evolutionary analysis revealed that NgR1 was genetically closer to human (Homo sapien) and monkey (Macaca) than to mouse (Mus musculus) and rat (Rattus rattus), as illustrated in Supplementary Fig. 3.
The tree shrew and human protein sequences were 90.34% identical. This was higher than Macaca fascicularis (89.91%), Macaca mulatta (89.91%), mouse (89.06%), and rat (88.84%).
The 3D structure of human and tree shrew NgR1 was created using the SWISS-MODEL integrated with visualization program PyMOL2.2.0 (DeLano Scientific, San Carlos, CA, USA). This was found to be similar between the two species. NgR1 has nine leucine-rich repeats (LRR), a C-terminal cap domain (LRRCT), and an N-terminal cap domain (LRRNT) (Fig. 1A,B). An alpha helix was present in the human LRRNT region and sixth LRR, but not in the tree shrew. The tree shrew NgR1 had an alpha helix at the LRRCT end, but not human NgR1 (Fig. 1C). These results show that tree shrew and human NgR1 have similar, but not identical structures (Fig. 1).
Predicted structure of NgR1 using the SWISS-MODEL integrated with visualization program PyMOL2.2.0. (A) Tree shrew NgR1 protein. Specific domains include nine LRR (red), a LRRNT (blue) and a LRRCT (yellow). (B) Human NgR1 protein. Specific domains include nine LRR (purple), a LRRNT (blue) and a LRRCT (yellow). (C) The superimposed conformation of human (yellow) and tree shrew (green) NgR1, differential amino acids shown in red.
qPCR was used to measure the mRNAs expression in 26 tissues from the tree shrew in order to study the NgR1 expression pattern. NgR1 mRNAs were mainly distributed in the central nervous system, especially in the temporal lobe, frontal lobe and parietal lobe (Fig. 2). However, the NgR1 mRNA level in the peripheral nervous system was relatively low. The tissue-specific expression pattern observed here for NgR1 suggests that it may play a role in neurodegenerative diseases and in neurotropic virus infection.
Expression pattern of NgR1 in the tree shrew. Gene-specific primers (Supplementary Table 2) were used to measure NgR1 mRNA expression in 26 tissues from the tree shrews using RT-PCR, with the tsGAPDH gene used for normalization.
To examine the function of NgR1 on infection by Mammalian Reovirus (MRV),
anti-Nogo receptor antibody (anti-NgR1), Neuraminidase (NA),
Phosphatidylinositol-specific phospholipase C (PI-PLC) and NEP1-40 were first
used to block NgR1. NEP1-40 is an NgR1 antagonist peptide comprised of 40 amino
acids from the N-terminal of Nogo-66. MRV1/TS/2011 was then incubated with tNC
Simultaneous addition of anti-NgR1 and NA significantly decreased the MRV1/TS/2011 copy in tNC compared to the addition of anti-NgR1, NA, PI-PLC or NEP1-40 (Fig. 3). Furthermore, the addition of anti-NgR1, NA, NEP1-40 or PI-PLC significantly reduced the MRV1/TS/2011 copy in tNC compared to cells treated by PBS. Hence, these four NgR1 blockers used alone or in combination can reduce MRV1/TS/2011 copy in tNC compared to cells treated by PBS.
Infection blockade of MRV1/TS/2011 in tNC. (A) The effect of
different treatments on infection of tNC cells by MRV1/TS/2011. Infection was
significantly reduced when anti-Nogo Receptor antibody (anti-NgR1) or
Neuraminidase (NA) was added individually, and almost completely blocked, when
anti-NgR1 and NA were added simultaneously. Infection was similarly reduced when
Phosphatidylinositol specific phospholipase C (PI-PLC) or NEP1-40 was added.
Simultaneous addition of PI-PLC and NA, or of NEP1-40 and NA, also reduced
infection more than the single agent. (B) Image Pro Plus software 6.0 was used to
evaluate the proportion of red fluorescence positive cells amongst the total
number of cells shown in Fig. 3A. Significant differences were scored as:
Nogo is part of the reticulon protein family and consists of three subtypes: Nogo-A, Nogo-B and Nogo-C. Each subtype has a common 66 amino acid domain (Nogo-66) that can mediate an inhibitory effect on axon growth through the Nogo receptor (NgR) [1, 2, 3, 30]. NgR is a leucine-rich repeat protein encoded by the RTN4R gene. In vivo, NgR is mainly expressed in muscle and neuronal cell bodies [5, 6, 7, 8].
Here, we describe the Nogo-66 receptor (NgR1) in the Chinese tree shrew. The tree shrew NgR1 is 3128 bp in length and the ORF sequence is 2193 bp, encoding 731 amino acids (Supplementary Fig. 1). In comparison, human NgR1 consists of 473 amino acid residues  and zebrafish NgR1 encodes 479 aa . According to the multiple sequence alignment, tree shrew NgR1 was more homologous to human (90.34%) than to mouse or rat (Supplementary Fig. 2). Previous reports showed that human homologous mouse NgR1 gene cDNA had 89% amino acid homology . Results from the phylogenetic tree showed that tree shrew NgR1 was genetically closer to human and monkey NgR1 than to rat and mouse (Supplementary Fig. 3). The NgR1 showed the same leucine-rich repeat (LRR) domain structure reported previously for human NgR1 [4, 31]. It is also capped by C-terminal and N-terminal cysteine-rich modules termed LRRCT and LRRNT segments, respectively (Fig. 1). However, an alpha helix is present in the human NgR1 LRRNT region and in the sixth LRR, and whereas the alpha helix in tree shrew NgR1 is present in the LRRCT end (Fig. 1C). Furthermore, the amino acid length of NgR1 is longer than human NgR1, while the 3D structure alignment starts from the 285th amino acid of tree shrew NgR1. Further studies are required to determine the possible impact of these differences on tree shrew NgR1 founction.
Previous studies showed that NgR was predominantly expressed in the adult brain, eye and heart, with low expression level in the spinal cord and gill [28, 32]. The expression pattern of NgR1 in the tree shrew is consistent with that reported in the human, mouse and zebrafish, with expression observed mainly in the CNS [3, 32, 33]. Therefore, the expression pattern of NgR1 in the tree shrew is similar to that of humans and other mammals, suggesting evolutionarily conserved functions.
In our previous studies we isolated two strains of MRV (MRV1/TS/2011 and MRV3/TS/2012) from the feces of tree shrews . Other researchers have reported that cells expressing NgR1 can mediate Mammalian Reovirus binding and infection of non-susceptible cells. NgR1-specific antibody can effectively block the binding of Mammalian Reovirus to cells that expressed NgR1 . In the present study, NgR1 blockers (anti-NgR1, NA, PI-PLC and NEP1-40), were used to inhibit the infection of reovirus in tree shrew primary neuronal cells (tNC). These four antagonists were observed to decrease MRV1/TS/2011 infectivity in tNC compared to cells treated with PBS. Furthermore, combined use of anti-NgR1 and NA significantly reduced MRV1/TS/2011 infectivity in tNC (Fig. 3). Our results indicate that NgR1 can mediate Mammalian Reovirus infection in tree shrew primary neuronal cells, but this can be blocked using NgR1 antagonists. These findings are consistent with those reported by Dermody .
In conclusion, we analyzed the structure and function of the NgR1 gene in the Chinese tree shrew. Current knowledge regarding NgR1 is still limited and further studies are needed to investigate other functions of this gene. For example, nerve cells from the temporal and occipital lobes could be isolated and used for MRV1/TS/2011 infection and blocking experiments in order to investigate whether infectivity is related to the expression of NgR1 in different parts of the brain.
CXL, XYK and XFL designed the study, performed bioinformatics analysis, done the qRT-PCR experiments and cell culture and also drafted the manuscript; WGW, XMS, NL and PFT supplied the animals and collected the tissues; JJD designed the study and revised the manuscript. All authors have read and approved the manuscript.
The research project was approved by the institutional Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences (DWSP201803034), and all the procedures were performed according to ethical standards and practices.
This work was mainly supported by the Yunnan health training project of high-level talents (D-2018026), Yunnan science and technology talent and platform program (2018HB071) and the Kunming Science and technology innovation team (2019-1-R-24483), the National Natural Science Foundation of China (No U1702282), Yunnan province Key Laboratory project (2017DG008).
The authors declare no conflict of interest.
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