NRP1 and Furin as Putative Mediators of SARS-CoV-2 Entry into Human Brain Cells⋆

Abstract

Background:

Coronavirus disease 2019 (COVID-19) has prominent neurological manifestations, including psychiatric symptoms, indicating a significant synaptic pathology. Surprisingly, evidence suggests that key severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host cell entry mediators angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) are negligibly expressed in the human brain, complicating the understanding of neuropsychiatric pathomechanisms manifestations in COVID-19. Recent studies have suggested that an alternative host cell entry receptor, neuropilin-1 (NRP1), can mediate the entry of furin-cleaved SARS-CoV-2 spike proteins into host cells. However, the role of NRP1 and furin in mediating SARS-CoV-2 entry into human brain cells has been the least explored and remains a lacuna in the literature.

Method:

We performed an in silico analysis of the transcriptomic and proteomic expressions of SARS-CoV-2 host-cell entry receptors (ACE2 and NRP1) and associated tissue proteases (TMPRSS2 and furin) in human brain tissue using the publically available databases.

Results:

ACE2 and TMPRSS2 were not detected in transcriptomic and proteomic expression analysis in any human brain regions. In contrast, transcriptomic and proteomic expressions of NRP1 and furin were detectable (N.X.≥ 1) across the human brain. The protein expressions of NRP1 and furin were observable in the brain tissue’s neuronal cells, neuropil, and glial cells.

Conclusion:

Based on the expression analysis and emerging evidence in the literature, we hypothesize that SARS-CoV-2 entry in human brain cells may be mediated through NRP1 and furin. Furthermore, invivo, experimental evidence will be needed to prove this hypothesis.

Introduction

Coronavirus disease 2019 (COVID-19) has prominent neurological manifestations as acute and long-term sequelae, including psychiatric symptoms.1,2 Psychiatric manifestations primarily have indicated significant synaptic pathology. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the viral agent of COVID-19, is known to enter host cells through a cell surface receptor, ACE2—angiotensin-converting enzyme 2.3 Along with ACE2, the cellular expressions of hostproteases such as transmembrane protease, serine 2 (TMPRSS2) and furin, which cleavethe viral spike protein, are considered essential for SARS-CoV-2 infection.3 SARS-CoV spike (S) protein uniquely contains a polybasic sequence motif, Arg-Arg-Ala-Arg, at the S1/S2 boundary (682–685 aa). It provides a cleavage site for the host serine protease, furin. Inclusion of the furin cleavage site (FCS) is an evolutionary gain in SARS-CoV-2, considering its conspicuous absence in other related SARS viruses, including SARS-CoV-1, and has been shown to increase infectivity and tissue tropism.3 The emerging SARS-CoV-2 variants have accumulated mutations at the FCS, potentially favoring furin-mediated processing.4 The furin-cleaved C-terminus of the S1 protein conforms to a unique peptide sequence—[R/K]XX[R/K] motif, termed as the “C-end rule,” which can potentially bind to neuropilin receptors expressed on the host cells. The “C-end rule” sequence is known to be present in a set of host peptides involved in pleiotropic physiological/pathological activities, such as axonal guidance, angiogenesis, vascular permeability, immune regulation, and survival of cancer cells.5 Recent studies suggested neuropilin-1 (NRP1) can bind to furin-cleaved SARS-CoV-2 spike protein and facilitate ACE2-mediated SARS-CoV-2 entry into host cells.6 Also, NRP1 can independently mediate viral entry into the host cells in higher viral titers.6,7 Notably, blocking the NRP1-spike protein interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture.6,7

Currently, the pathomechanisms of neuropsychiatric manifestations in COVID-19 remain the least understood. Among diverse contenders, a receptor-based mediation of SARS-CoV-2 entry in the human brain, either through olfactory or vagal nerves or neurovascular routes, remains a plausible mechanism.8 However, the role of ACE2 in mediating SARS-CoV-2 entry into brain cells appears less plausible as existing evidence suggests that there is negligible ACE2 expression in human brain tissue.8 Therefore, there is a need to look for an alternative cell receptor that can mediate SARS-CoV-2 entry into the human brain in the absence of ACE2. Finding the key mediators of SARS-CoV-2 entry into the brain cells remains a lacuna in the literature. Multiple authors have suggested an NRP1-mediated mechanism behind neuropsychiatric symptoms in COVID-19 since identifying the NRP1 as an alternative SARS-CoV-2 entry receptor in human cells.7,8 However, the studies that have studied relative distributions for the SARS-CoV-2 entry mediators and entry-associated molecules in the human brain are lacking. In this study, we performed an in silico analysis of the publically available databases providing transcriptomic and proteomic expressions of ACE2 and TMPRSS2, and NRP1 and furin in human brain regions. This study aimed to know whether NRP1 and furin may have an edge in mediating COVID-19 neuropathology compared to ACE2 and TMPRSS2.

Methods

We performed in silico analysis of mRNA and protein expressions of ACE2, NRP1, TMPRSS2, and furin in human brain immune components using the tissue transcriptome and immunohistochemistry (IHC) data available in Human Brain Atlas, a subsection of Human Protein Atlas (https://www.proteinatlas.org/).

External data source methods (as described by the source labs)

The source laboratory estimated mRNA expression and localization of human proteins using deep sequencing of RNA (RNA-seq) and IHC in normal tissue.

IHC

The specimens containing normal tissue were collected per approval from the local ethics committee. The specimens were derived from surgically removed normal tissue. Normal was defined by tissue-specific morphological parameters and the absence of neoplasia. Antibodies against human ACE2 (HPA000288 and CAB026174), NRP1 (HPA030278 and CAB004511), TMPRSS2 (HPA035787), and furin (CAB009499) were used. IHC staining was performed on normal tissue microarray using a standard protocol (https://www.proteinatlas.org/download/IHC_protocol.pdf). The protein expression score (low, medium, and high) for each protein was calculated based on the staining intensity (negative, weak, moderate, or strong) and the fraction of stained cells (<25%, 25–75%, or >75%).

Human brain transcriptomics

The transcriptomic data were collected from the three databases (HPA, GTEx, and FANTOM5). For HPA RNAseq, total RNA was extracted from the tissue samples of healthy individuals (accession no: PRJEB4337, Ensembl: ENSG00000130234 (version 92.38)) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) with the standard-sensitivity RNA chip or an Agilent 2100 Bioanalyzer system with the RNA 6000 Nano Labchip Kit (Agilent Biotechnologies, Palo Alto, USA) was used to analyze the extracted RNA samples. Only the samples of RNA Integrity Number > 7.5 were used for the mRNA sequencing. The mRNA sequencing (read length of 2 × 100 bases) was performed using Illumina HiSeq2000 and 2500 machines (Illumina, San Diego, CA, USA) as per the standard Illumina RNA-seq protocol. Kallisto v0.43.1 (https://pachterlab.github.io/kallisto/about) was used to estimate the transcript abundance. Furthermore, the normalized tags per million for each gene from the three screened databases were calculated. Each tissue was categorized for the intensity of gene expression using a cutoff value of 1 N.X. (as a limit for detection across all tissues). The transcriptomic expression of an analyzed protein in a particular tissue type was categorized as (i) enriched, if it had an N.X. level at least four times higher than other tissues; (ii) low specificity, if N.X.≥ 1 in at least one tissue; (iii) not detected: if N.X. < 1 in all tissues.

Meta-data analysis

We analyzed HPA transcriptomic and proteomic data regarding tissue-specific expression of the screened proteins in human brain components. We matched the IHC staining profile with mRNA expression data for each protein to yield an “annotated protein expression profile.” Graphs were plotted, and inferences were made based on the final results.

Results and Discussion

The pathogenesis of the neuropsychiatric manifestations of COVID-19 remains little understood. SARS-CoV-2 structural proteins and genomic components have often been detected in the autopsied brain tissue of COVID-19 patients indicating direct neuroinvasion.9,10 However, there is an extreme lack of knowledge about the molecular mediators of SARS-CoV-2 entry into the brain cells. Finding the key mediators of SARS-CoV-2 entry into the brain cells may help develop target-based therapeutic strategies and suitable drug targets for treating neuro-COVID. Our study unravels relative expressions of SARS-CoV-2 host cell-entry receptors ACE2 and NRP1 and entry-associated host proteases TMPRSS2 and furin in human brain regions providing essential insights regarding the viral entry into the brain cells and consequent neuropathology. ACE2 and TMPRSS2 were not-detected in transcriptomic (N.X. < 1) and proteomic expression analysis in any of the human brain regions, including hippocampal formation (Figs.1a and 2). In contrast, transcriptomic and proteomic expressions of NRP1 and furin were detectable (N.X. ≥ 1) across the human brain (Figs.1b and 2). The protein expressions of NRP1 and furin were observable in the brain tissue’s neuronal cells, neuropil, and glial cells (Fig.3).

FIG. 1.

Transcriptomic expression of SARS-CoV-2 entry factors in different parts of the brain. (a) ACE2 and TMPRSS2; (b) NRP1 and furin. Data source: Human Protein Atlas, https://www.proteinatlas.org/.

FIG. 2.

Proteomic expression of SARS-CoV-2 entry factors in different parts of the brain. NC: neuronal cells; GC: glial cells; EC: endothelial cells; GL: granular layer; ML: cells of molecular layer; PC: Purkinje cells; NA: data not available. Software: Human Protein Atlas, https://www.proteinatlas.org/.

FIG. 3.

Immunohistochemical expression in the human cerebral cortex (a) NRP1, (b) furin. Arrows indicate immunostaining in the cytoplasmic membrane of neuronal cells. In addition, moderate to intense staining can also be noted in the neuropil (intercellular area) and cytoplasmic membrane of glial cells (with small blue nuclei). Data source: Human Protein Atlas, https://www.proteinatlas.org/.

Substantial histopathological evidence has been presented by recent studies for the direct viral invasion of the human brain cells in COVID-19, at least in a particular proportion of cases.6,7,10,11 Of note, the canonical way of SARS-CoV-2 cell entry depends on the co-localized expression of ACE2 and TMPRSS2.3 In contrast, we observed in our analysis that ACE2 and TMPRSS2 were undetectable in the human brain tissue; however, NRP1 and furin were co-expressed in abundance across the brain regions.

There has been robust evidence for the SARS-CoV-2 infection of the olfactory epithelial cells in COVID-19 patients,9 which is considered a potential route for viral entry into the brain.8 Interestingly, the viral infection of the olfactory epithelium was shown to localize to the NRP1-positive cells in the landmark study by Cantuti-Castelvetri etal.6 The authors observed high immunoreactivity for NRP1; on the contrary, they observed scarcely detectable ACE2 in the epithelial surface layer of the human olfactory epithelium in autopsy tissue samples. NRP1 was also observed in oligodendrocyte transcription factor 2 (OLIG2) cells, commonly expressed by olfactory neuronal progenitors. The authors showed in the tissue samples from autopsied COVID-19 patients and noninfected control patients that SARS-CoV-2 could infect NRP1-positive cells. The infected olfactory epithelial cells (showing positive staining for antibodies against spike protein) showed high expression of NRP1 with a co-staining for OLIG2.6

Our study unravels significant expression of NRP1 and furin in neuronal and neuroglial cells and associated neuropil, including within the olfactory region and hippocampal formation (Figs.1 and 2). The emerging evidence for the NRP1-mediated SARS-CoV-2 neuroinvasion11 suggests that it may have a role in the pathomechanisms of synaptic pathologies, such as loss of the sense of smell and taste, memory loss, and multiple psychiatric manifestations in COVID-19, as the acute or long-term sequelae.12 –15

The most crucial postmortem evidence that NRP1 can mediate SARS-CoV-2 infection of the brain cells was presented in a recent study by Crunfli etal.11 The authors showed that SARS-CoV-2 primarily infected astrocytic glial cells (65.93%) in the human brain cell culture, which was NRP1 mediated. They also detected SARS-CoV-2 spike protein in neurons (NeuN+ cells), however, to a limited extent. They further showed the viral infection of the astrocytes induced changes in the metabolism of key proteins and metabolites used to fuel neurons and in the biogenesis of neurotransmitters, which can reasonably explain the COVID-19 neuropathology.11

Notably, NRP1 also has a known role in the regeneration and plasticity of adult neurons.16 It works as a receptor for Semaphorin 3A—an axonal guidance protein16 and vascular endothelial growth factor A (VEGF-A)¹⁷—a key molecule involved in hippocampal neurogenesis. A recent study has shown that the SARS-CoV-2 spike protein binding to NRP1 in sensory nerves can induce analgesia by altering VEGF-A-mediated neuronal signaling.17 Of note, the olfactory epithelium and the hippocampus are the known sites for ongoing neurogenesis in adult humans; interestingly, both brain regions are also implicated in COVID-19 neuropathology. ASARS-CoV-2 infection-induced disruption of the ongoing olfactory18,19 and hippocampal20 neurogenesis and plasticity in COVID-19 patients remains plausible, given high NRP1 and furin expressions in these brain regions. A recent study by Bayat etal. in postmortem specimens from COVID-19 patients provided robust evidence for disrupted hippocampal neurogenesis.21 Disrupted neurogenesis can also explain frequent olfactory and hippocampal dysfunctions in COVID-19 cases.18,20 Furthermore, the SARS-CoV-2-induced neuro-immune dysfunction and neuro-inflammation can also be mediated through NRP1, given its known role as an immune checkpoint of T cell memory and inflammation.22

Thus, synthesizing the evidence from ours and existing studies makes a strong case for the NRP1-furin-based model of neuro-COVID-19. On the contrary, some authors have suggested systemic inflammation and immune activation-based origin of the neuropsychiatric symptoms in the absence of significant expressions of canonical SARS-CoV-2 cell entry mediators.23,24 However, concrete evidence is still scarce to support this alternative pathogenesis model that excludes direct neuroinvasion.9,10

In contradiction to our findings, some studies reported varying extents of transcriptomic expression of ACE2 and TMPRSS2 in human brain cells.25 –27 However, there is a considerable lack of corroborative evidence for the significant proteomic expression of these molecules in human brain cells.28 –30 Thus, their substantial contribution in mediating SARS-CoV-2 entry into human brain cells, specifically the neurons, remains doubtful.

Furthermore, some authors also proposed the possible role of CD147, an alternative receptor for SARS-CoV-2 entry in human cells,31 in COVID-19 neuropathology.25 However, available evidence suggests that proteomic expression of this molecule in the human brain is limited to the endothelium of the vessels and neuropils; hence, its role in the direct viral invasion of the brain cells seems less likely.32

Conclusions and Further Directions

The transnasal inoculation of the virus in transgenic animals expressing human versions of the SARS-CoV-2 entry molecules8,33 can be used for preparing neuro-COVID-19 pathogenesis models. The human brain organoids also can be a promising option for this purpose.34 The studies employing such models can examine the differential role of NRP1 and furin in mediating SARS-CoV-2 cell entry in the presence and absence of canonical SARS-CoV-2 cell entry receptors.

Limitations

For certain brain regions, proteomic expression data were unavailable to corroborate the studied markers’ transcriptomic expressions.

Conflict of Interest: All the authors declare “No Conflict of Interest.”

Author Contributions: A.K. and V.P. conceived the idea. A.K., R.K.N., and P.P. analyzed the data and prepared figures/graphs. A.K. wrote the first draft. S.K., R.K.N., C.K., V.P., R.K.J., and P.P. reviewed and edited for the final draft.

Funding: There was no dedicated funding for this project.

Data Availability: Data used for this study can be accessed at the following link: https://www.proteinatlas.org/about/download.

Footnotes

Acknowledgments

The authors acknowledge The Human Protein Atlas (https://www.proteinatlas.org/) for providing relevant data in the public domain. This manuscript has been released as a preprint at BioRxiv ().

References

Taquet

, Geddes

, Husain

, et al. 6-Month neurological and psychiatric outcomes in 236 379 survivors of COVID-19: A retrospective cohort study using electronic health records. Lancet Psychiatr. 2021; 8(5): 416–427. DOI: 10.1016/S2215-0366(21)00084-5

Varatharaj

, Thomas

, Ellul

, et al. Neurological and neuropsychiatric complications of COVID-19 in 153 patients: A UK-wide surveillance study. Lancet Psychiatr. 2020; 7: 875–882. DOI: 10.1016/s2215-0366(20)30287-x

Kumar

, Prasoon

, Kumari

, et al. SARS-CoV-2-specific virulence factors in COVID-19. J Med Virol. 2021; 93(3): 1343–1350. DOI: 10.1002/jmv.26615

Kumar

, Parashar

, Kumar

, et al. Emerging SARS-CoV-2 variants can potentially break set epidemiological barriers in COVID-19. J Med Virol. 2022; 94(4): 1300–1314. DOI: 10.1002/jmv.27467

Mayi

, Leibowitz

, Woods

, et al. The role of neuropilin-1 in COVID-19. PLOS Pathog. 2021; 17(1): e1009153. DOI: 10.1371/journal.ppat.1009153

Cantuti-Castelvetri

, Ojha

, Pedro

, et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Science. 2020; 370(6518): eabd2985–860. DOI: 10.1126/science.abd2985

Daly

, Simonetti

, Klein

, et al. Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science. 2020; 370(6518): 861–865. DOI: 10.1126/science.abd3072

Kumar

, Pareek

, Prasoon

, et al. Possible routes of SARS-CoV-2 invasion in brain: In context of neurological symptoms in COVID-19 patients. J Neurosci Res. 2020; DOI: 10.1002/jnr.24717

Meinhardt

, Radke

, Dittmayer

, et al. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat Neurosci. 2021; 24(2): 168–175. DOI: 10.1038/s41593-020-00758-5

10.

Serrano

, Walker

, Arce

, et al. Mapping of SARS-CoV-2 brain invasion and histopathology in COVID-19 disease. medRxiv. 2021; DOI: 10.1101/2021.02.15.21251511

11.

Crunfli

, Carregari

, Veras

, et al. Morphological, cellular, and molecular basis of brain infection in COVID-19 patients. Proc Natl Acad Sci USA. 2022; 119(35): e2200960119. DOI: 10.1073/pnas.2200960119

12.

Arunachalam

, Anoop

, Verma

, et al. SARS-CoV-2: The road less traveled: The respiratory mucosa to the brain. ACS Omega. 2021; 6(10): 7068–7072. DOI: 10.1021/acsomega.1c00030

13.

Chapoval

, Keegan

AD.

Perspectives and potential approaches for targeting neuropilin 1 in SARS-CoV-2 infection. Mol Med. 2021; 27(1): 1–15. DOI: 10.1186/s10020-021-00423-y

14.

Mone

, Gambardella

, Wang

, et al. miR-24 targets the transmembrane glycoprotein neuropilin-1 in human brain microvascular endothelial cells. ncRNA. 2021; 7(1): 9. DOI: 10.3390/ncrna7010009

15.

Zalpoor

, Shapourian

, Akbari

, et al. Increased neuropilin-1 expression by COVID-19: A possible cause of long-term neurological complications and progression of primary brain tumors. Hum Cell. 2022; 35(4): 1301–1303. DOI: 10.1007/s13577-022-00716-2

16.

Gavazzi

Semaphorin-neuropilin-1 interactions in plasticity and regeneration of adult neurons. Cell Tissue Res. 2001; 305(2): 275–284. DOI: 10.1007/s004410100365

17.

Moutal

, Martin

, Boinon

, et al. SARS-CoV-2 spike protein co-opts VEGF-A/neuropilin-1 receptor signaling to induce analgesia. Pain. 2021; 162(1): 243–252. DOI: 10.1097/j.pain.0000000000002097

18.

Rethinavel

, Ravichandran

, Radhakrishnan

, et al. COVID-19 and Parkinson’s disease: Defects in neurogenesis as the potential cause of olfactory system impairments and anosmia. J Chem Neuroanat. 2021; 115: 101965. DOI: 10.1016/j.jchemneu.2021.101965

19.

Butowt

, von Bartheld

CS.

Anosmia in COVID-19: Underlying mechanisms and assessment of an olfactory route to brain infection. Neuroscientist. 2021; 27(6): 582–603. DOI: 10.1177/1073858420956905

20.

Klein

, Soung

, Sissoko

, et al. COVID-19 induces neuroinflammation and loss of hippocampal neurogenesis. Res Sq. 2021; 29: 29. DOI: 10.21203/RS.3.RS-1031824/V1

21.

Bayat

, Azimi

, Hassani Moghaddam

, et al. COVID-19 causes neuronal degeneration and reduces neurogenesis in human hippocampus. Apoptosis. 2022; 1: 1–17. DOI: 10.1007/s10495-022-01754-9

22.

Liu

, Somasundaram

, Manne

, et al. Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity. Nat Immunol. 2020; 21(9): 1010–1021. DOI: 10.1038/s41590-020-0733-2

23.

Boldrini

, Canoll

, Klein

RS.

How COVID-19 affects the brain. JAMAPsychiatry. 2021; 78(6): 682–683. DOI: 10.1001/jamapsychiatry.2021.0500

24.

Spudich

, Nath

Nervous system consequences of COVID-19. Science. 2022; 375(6578): 267–269. DOI: 10.1126/science.abm2052

25.

Qiao

, Li

, Bao

, et al. The expression of SARS-CoV-2 receptor ACE2 and CD147, and protease TMPRSS2 in human and mouse brain cells and mouse brain tissues. Biochem Biophys Res Commun. 2020; 533(4): 867–871. DOI: 10.1016/j.bbrc.2020.09.042

26.

Wang

, Xu

scRNA-seq profiling of human testes reveals the presence of the ACE2 receptor, a target for SARS-CoV-2 infection in spermatogonia, leydig and sertoli cells. Cells. 2020; 9(4): 920. DOI: 10.3390/cells9040920

27.

Chen

, Wang

, Yu

, et al. The spatial and cell-type distribution ofSARS-CoV-2 receptor ACE2 in the human and mouse brains. FrontNeurol. 2021; 11: 1860. DOI: 10.3389/FNEUR.2020.573095/BIBTEX

28.

Hamming

, Timens

, Bulthuis

MLC

, et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004; 203(2): 631–637. DOI: 10.1002/path.1570

29.

Hikmet

, Méar

, Edvinsson

, et al. The protein expression profile of ACE2 in human tissues. Mol Syst Biol. 2020; 16(7): 9610. DOI: 10.15252/MSB.20209610

30.

Lindskog

, Méar

, Virhammar

, et al. Protein expression profile of ACE2 in the normal and COVID-19-affected human brain. J Proteome Res. 2022; DOI: 10.1021/ACS.JPROTEOME.2C00184

31.

Wang

, Chen

, Zhang

, et al. CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells. Signal Transduct Target Ther. 2020; 5(1): 1–10. DOI: 10.1038/s41392-020-00426-x

32.

The Human Protein Atlas: Tissue expression of BSG—Summary. Available at https://www.proteinatlas.org/ENSG00000172270-BSG/tissue. Accessed September 10, 2022.

33.

Park

, Pino

, Akhter

, et al. Animal models of COVID-19: Transgenic mouse model. Methods Mol Biol. 2022; 2452: 259–289. DOI: 10.1007/978-1-0716-2111-0_16

34.

Ramani

, Pranty

, Gopalakrishnan

Neurotropic effects of SARS-CoV-2 modeled by the human brain organoids. Stem Cell Rep. 2021; 16(3): 373–384. DOI: 10.1016/j.stemcr.2021.02.007