This study analyzed and compared the DEGs related to lung epithelial cell lines (NHBE and A549) treated with SARS-CoV-2 and N-Tera2 differentiated human neuronal cells treated with HCoV-OC43 to investigate the possible molecular mechanisms behind the neurological manifestations of SARS-CoV-2 infection. We constructed TF-miR-gene regulatory and protein-protein interaction networks to identify the critical nodes (hubs, bottlenecks, motif members, and MCODE cluster members), biological processes, and pathways mediating in the two infections' pathogenesis. The shared critical genes between the HCoV-OC43 effect on neural cells and SARS-CoV-2 could shed light on the molecular mechanisms of brain conditions in COVID-19 patients (fig.9). Herein, we discuss and validate some of the predicted genes and pathways probably mediating in neural manifestations of COVID-19 using other experimental literature.
This study investigated the genes mediating in the central nerve system (CNS) involvement of SARS-CoV-2 using the shared genes between both OC43 and SARS-CoV-2 GRN and PPI networks. The most critical genes included six up-regulated shared genes (IL6, TNF, HOXA5, POU2F2, ITGB3, and STAT1) and 12 down-regulated shared genes (YY1, E2F6, ESR1, FOXO3, FOXO1, MEF2A, ATF3, ATF4, DDIT3, TCF4, BCL2L2, and BMP4). Several other studies have previously reported the mentioned critical genes to be involved in neural proliferation and differentiation (neurodevelopment), neurotransmission, synaptic plasticity, and myelination (Each molecule is separately described and referred to in table 9) (Yang, Lindholm et al. 2002, Nakanishi, Niidome et al. 2007, Ragel, Couldwell et al. 2007, He and Casaccia-Bonnefil 2008, Imamura, Satoh et al. 2008, Lange, Chavez et al. 2008, Islam, Gong et al. 2009, Renault, Rafalski et al. 2009, Leung and Cahill 2010, Oh, McCloskey et al. 2010, Zolova and Wight 2011, Hunt, Raivich et al. 2012, Pozo, Cingolani et al. 2012, Cosker, Pazyra-Murphy et al. 2013, Wang, Choi et al. 2013, Ma, Tang et al. 2014, Mazalouskas, Jessen et al. 2015, Varney, Polston et al. 2015, Doan, Kinyua et al. 2016, Kennedy, Rahn et al. 2016, Chen, Gao et al. 2017, Lizen, Moens et al. 2017, Higashi, Tanaka et al. 2018, Li, Jin et al. 2018, Liu, Amar et al. 2018, Zhu, Carmichael et al. 2018, Liu, Yu et al. 2019, Majidi, Reddy et al. 2019, Masgutova, Harris et al. 2019, Wu and Donohoe 2019, Hartman and Czyz 2020, Pennycook, Vesela et al. 2020). Below, we have hypothesized how some of these in-silico identified critical genes can play roles in neural manifestations of COVID-19 pathogenesis.
The SARS-CoV-2 RNA acts as a viral pathogen-associated molecular pattern (PAMPs) that can be identified by pattern-recognition receptors (PRRs) like toll-like receptors (TLRs) and NOD (nucleotide-binding oligomerization domain)-like receptors (NLRs) (Kopitar-Jerala 2015). The surface receptors were among the top enrichment results of KEGG pathways of the shared DEGs between the two infections and are the first molecules activated in the innate immune system against the CNS pathogens. An inflammatory cascade initiates after triggering TLRs 3, 7, 8, and 9 by activating the NF-kB signaling pathway and IFN α/β/γ gene expression (Sabroe, Parker et al. 2008, Totura, Whitmore et al. 2015). The NF-kB pathway was also another pathway enriched for the shared DEGs. It regulates gene expression by kB sites present in promoter and enhancer regions of various essential genes such as chemokines, cytokines, adhesion molecules, and pro-inflammatory transcription factors. Therefore, it regulates neuronal survival and neuronal inflammatory reactions. The NF-κB also induces pro-IL-1β, pro-IL-18, TNF, and IL-6 (Tergaonkar, Correa et al. 2005, Tergaonkar 2006, Wong and Tergaonkar 2009). The TNF and IL-6 were identified as hub/bottlenecks in the GRN/ PPI networks of our in-silico analysis.
SARS-CoV-2 can also be recognized by NLRP3 (a kind of NLRs). NLRP3 forms an inflammasome complex with caspase-1 and cleaves IL-1β and IL-18 to mature forms (Zhao and Zhao 2020). These cytokines (IL-1β, IL-18, TNF, and IL-6) induce further NF-κB nuclear translocation, activation of the JAK/STAT pathway, and phosphorylation of p38 MAPK (Battagello, Dragunas et al. 2020). The pathways were also included among our top ten enrichment results of the shared genes. Activation of p38s regulates immune response and inflammatory processes in SARS-CoV-2 infection (Feng, Fang et al. 2019, Grimes and Grimes 2020). In CNS, elevated activity of MAPK signaling can modulate neuronal survival and homeostasis (Feng, Fang et al. 2019, Grimes and Grimes 2020). Besides, in the JAK/STAT pathway, cytokines like INF and IL-6 bind to their receptors, induce JAK proteins cross-phosphorylation and then recruit STAT proteins. The phosphorylated STAT proteins then dimerize and translocate into the nucleus and regulate gene expression as transcription factors and (O'Shea, Schwartz et al. 2015). STAT1 and STAT3 were also identified as two crucial genes in our GRN/PPI networks. STAT1 has an essential role in the IFN signaling type I and type II and the JAK/STAT pathway (Pasieka, Cilloniz et al. 2011, Kulkarni, Scully et al. 2017). The role of STAT-1 has been previously elucidated in the innate immune response to other neurotropic viruses such as the severe acute respiratory syndrome coronavirus (SARS), Herpes simplex virus type 1 (HSV-1), and West Nile virus (WNV) (Pasieka, Cilloniz et al. 2011, Mahlakõiv, Ritz et al. 2012, Winkelmann, Luo et al. 2016).
The neurotrophin signaling pathway was another enriched result between the shared genes of our in-silico study. The pathway makes crosslinks with various intracellular signaling cascades, including NF- kB and MAPK pathways. Neurotrophins, such as nerve growth and brain-derived neurotrophic factors, induce neurons' survival, development, and function (Reichardt 2006).
In the nervous system, IL-6 and TNF-α are typically expressed at relatively low levels. However, their expression is up-regulated under different pathological conditions like inflammation and viral infections (Yang, Lindholm et al. 2002, Oh, McCloskey et al. 2010). SARS-CoV-2 can activate glial cells in the CNS and induce a pro-inflammatory state. IL-6 and TNF-α play essential roles in the Systemic Inflammatory Response Syndrome (SIRS), leading to brain damage (Li, Fu et al. 2004, Liguori, Pierantozzi et al. 2020, Serrano-Castro, Estivill-Torrús et al. 2020, Wan, Yi et al. 2020). The IL-6 level also increases in other viral respiratory infections with neurological complications such as a human respiratory syncytial virus (RSV) and Influenza. It can be considered as an indicator of their neurologic prognosis (Aiba, Mochizuki et al. 2001, Kawashima, Kashiwagi et al. 2012, Morichi, Morishita et al. 2017).
HOXA5 was identified as another critical gene in our in-silico analysis. It is a member of the HOX family of transcription factors expressed throughout adulthood, especially in glutamatergic and GABAergic neurons. It regulates many genes associated with neuronal survival and synaptic function (Lizen, Moens et al. 2017). HoxA5 is reported to regulate the viral immediate-early (IE) gene expression in herpes simplex virus (HSV). Besides, the IE gene has an essential role in acute viral replication and its latency in neurons (Mitchell, De Santo et al. 1993, Mitchell 1995). HOXA5 expression is also reported to change significantly in some other neurotrophic viral infections like Cytomegalovirus, Coxsackievirus B3 (CVB3), and lymphocytic choriomeningitis virus (LCMV). The viruses can infect neurons and cause meningitis and encephalitis (Ester 2011, Puccini, Ruller et al. 2014).
POU2F2 (Oct-2) was another identified crucial gene. It has an essential role in virus replication and is a member of the POU family. It is predominantly expressed in B cells, activated T cells, and the nervous system (Luchina, Krivega et al. 2003). Some POU family members, such as POU2F1, are reported to be necessary for viral DNA replication and gene expression in other viruses such as herpes simplex virus (HSV) (Ryan and Rosenfeld 1997). It can be hypothesized that SARS-CoV-2 elevates the expression of HOXA5 and POU2F1 to increase its viral proliferation possibly.
The ITGB3 gene, coding for the integrin β3 subunit, is expressed and enriched at cortical, hippocampal, and midbrain synapses in the brain (Varney, Polston et al. 2015). The integrin β3 mediates in HSV-1 cell entry by relocating HSV receptor nectin1, and thus HSV to cholesterol-rich microdomains of the membrane where TLR2 presents. Therefore, integrin β3 plays a vital role in endocytosis of the virus and initiation of the innate immune response (NF-κB activation and production of IFNα, IFNβ, IL2, and IL10) (Gianni, Leoni et al. 2012, Gianni, Leoni et al. 2013). The ITGB3 was up-regulated in our in-silico results. Therefore, we suggest that SARS-CoV-2 is probably utilizing this upregulation to increase its entry. However, further experimental studies are required to confirm the prediction.
The antiviral response is also activated by Yin Yang 1 (YY1). It is a multi-functional transcription factor that can activate or repress gene expression in various cell types, including neurons (He and Casaccia-Bonnefil 2008, Chen and Chan 2019). Some Viral infections down-regulate the expression of YY1 since it can mediate the antiviral innate immune response and regulate the production of interferon-beta (IFN-β) (Zan, Zhang et al. 2017). YY1-1 can also repress the transcription of many retroviruses such as human immunodeficiency virus type I (HIV-1). It also contributes to a neurological disorder caused by the human T lymphotropic virus type 1 (HTLV-1) (Coull, Romerio et al. 2000, Wang and Goff 2020). We also identified the YY1 as a downregulated gene in SARS-CoV-2 and OC-43 infections. Therefore, it can be postulated that the virus is probably benefiting from the YY1-1 downregulation in its neural pathogenesis in COVID-19. E2F6 expression is a known mechanism that slows down or exits the cells from S-phase. Some viral proteins can inactivate E2F6 to extend the S-phase in virus-infected cells, such as human papillomavirus (HPV) E7 proteins, simian virus 40 T antigen, and adenovirus E1A (McLaughlin-Drubin, Huh et al. 2008). Our results showed that SARS-CoV-2 infection down-regulates the expression of E2F6. The E2F6 downregulation presumably contributes to the replication of the virus.
The Forkhead box O transcription factors (FOXO1 and 3) were the next identified critical genes. They mediate the regulation of the cell cycle, apoptosis, autophagy, and DNA repair. They also are reported to regulate neural cell survival, neuronal signaling, and stress responses in the nervous system (Santo and Paik 2018, Schäffner, Minakaki et al. 2018). Our results showed that SARS-CoV-2 down-regulated the FOXO proteins similar to other viruses such as the Japanese encephalitis virus (JEV). JEV induces cell apoptosis in neurons by inhibiting the FOXO-signaling pathway. Therefore, it can lead to severe viral encephalitis in humans and other animals (Guo, Yu et al. 2018). Furthermore, FOXO proteins have a validated role in the pathogenesis of HIV-I-infection and its associated neurological complications (Cui, Huang et al. 2009).
ESR1 (Estrogen receptor α) is present in the hypothalamus and amygdala regions related to human emotion and cognitive functions (Ma, Tang et al. 2014). It is also involved in the pathogenesis of hepatitis viruses (HBV & HCV) and their complications (Deng, Zhou et al. 2004, Zhai, Zhou et al. 2006, Watashi, Inoue et al. 2007). Neurological complications of these viruses range from peripheral neuropathy to cognitive impairment (Mathew, Faheem et al. 2016). SARS-CoV-2 is also a positive-sense RNA virus similar to HCV. Therefore, we predict that the molecular mechanisms behind the two viruses' similar neurological complications are probably the same, and we recommend the prediction for further experimental investigations.
Myocyte enhancer factor 2 (MEF2, isoform A) is also highly expressed in the brain. MEF2A is a downstream protein of NMDA receptor-mediated excitotoxicity in which excessive stimulation of the NMDA receptor contributes to the death of neurons. It is also reported to mediate in the neuropathogenesis of some other single-strand RNA-viruses such as HIV-1 (Kaul and Lipton 2004, Yndart, Kaushik et al. 2015). The MEF2A was identified as a critical gene and is recommended for further studies in the neuropathogenesis of SARS-CoV-2, similar to HIV-1.
Activating transcription factors (ATFs) are members of the ATF/cAMP response element-binding protein (CREB) family. ATF3 and ATF4 usually are low in neural cells, but their expression increases rapidly in response to pathological stress (Lange, Chavez et al. 2008, Hunt, Raivich et al. 2012). Activation of innate and adaptive immune systems can induce the expression of ATF3. However, ATF3 acts as a negative regulator of the immune response (Hunt, Raivich et al. 2012). Our analysis showed that SARS-CoV-2 down-regulated the ATF3. Therefore, it can be suggested that down-regulation of ATF3 possibly mediates in hyper-activation of the immune system and neural manifestations of some patients with severe COVID-19. ATF4 is a downstream protein of a cellular protective signaling pathway called the Unfolded Protein Response (UPR). UPR and Autophagy pathways are tightly interconnected and are reported to play a vital role in some viral infections such as hepatitis C virus, herpes simplex virus type 1 (HSV-1), Influenza virus, and severe acute respiratory syndrome (SARS) (Tardif, Mori et al. 2004, Versteeg, van de Nes et al. 2007, Burnett, Audas et al. 2012, Sen, Balakrishnan et al. 2014, Sims and Meares 2019). We hypothesize that SARS-CoV-2 probably modulates the UPR/Autophagy signaling pathways to produce large quantities of viral proteins. Besides, ATF4 (CERB-2) interacts with Human T-cell leukemia virus type 1 (HTLV-1) tax protein to regulate its transcription. The virus causes a severe neurologic disorder called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Ahuja, Kampani et al. 2006, Barbeau and Mesnard 2011).
DDIT3 (DNA damage-inducible transcript 3) was another down-regulated critical gene in our in-silico results. It is a downstream protein of ATF4 in unfolded protein response (UPR), which can activate the autophagy pathway (Bello-Perez, Sola et al. 2020). DDIT3 also downregulates BCL2, which is involved in the apoptosis pathway and autophagy flux (Nabirotchkin 2020). Therefore, SARS-CoV-2, similar to other previously known coronaviruses, dysregulates these three genes (ATF4, DDIT3, BCL2) to possibly exploit the autophagy molecular machinery and replicate more rapidly (Bello-Perez, Sola et al. 2020, Mamoor 2020, Nabirotchkin 2020). DDIT3 is also dysregulated in other viral infections such as the Zika virus (ZIKV), Tick-borne encephalitis virus (TBEV), and West Nile virus (WNV). The three viral infections can progress to the CNS and cause encephalitis or meningitis in severe cases (Selinger, Wilkie et al. 2017, Huang, West et al. 2019, Ojha, Rodriguez et al. 2019, Bonenfant, Meng et al. 2020).
Bcl2l2 (Bcl-w) is an Anti-apoptotic member of The B-cell lymphoma-2 (BCL-2) family (Hartman and Czyz 2020). Various viruses are reported to interact with the Bcl-2 family to regulate the cellular apoptosis, including Influenza A virus, hepatitis B virus, hepatitis C virus, Epstein–Barr virus, vesicular stomatitis virus, human immunodeficiency virus, and SARS-CoV. (Tan, Tan et al. 2007, Nencioni, De Chiara et al. 2009, Pearce and Lyles 2009, Busca, Saxena et al. 2012, Geng, Huang et al. 2012, Park, Kang et al. 2012, Ghigna, Reineke et al. 2013). Some of the viruses have previously been reported with neural complications. The Influenza A virus and SARS-CoV are reported to suppress BCL-W and Bcl-XL (another anti-apoptotic member of the family) to induce cellular apoptosis and subsequent tissue damage (Tan, Tan et al. 2007, Nencioni, De Chiara et al. 2009, Guan, Shi et al. 2012). Our results showed that SARS-CoV-2 also down-regulates Bcl-w, Similar to SARS-CoV. The Bcl-w probably also mediates in SARS-CoV-2 subsequent tissue damage in a similar way.
BMP4 is one of the Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor-beta (TGF-b) superfamily (Higashi, Tanaka et al. 2018). TGF-beta and BMP signaling pathways are activated following some neurotropic viral infections, such as reovirus. The signaling pathways are part of the host immune response and have a neuroprotective effect (Beckham, Tuttle et al. 2009). Besides, the synergistic activation of BMP and alpha-interferon signaling pathways is also reported to reduce the hepatitis C virus [136]. Based on our results, the SARS-CoV-2 down-regulated the BMP4. Therefore, we suggest that the virus probably is benefitted from the BMP4 down-regulation in infected neurons.
In this study, we investigated our 259 repurposed medicines indications and mechanisms using the DrugBank database. Our emphasis was on drugs that affect the nervous system; since SARS-CoV-2 is a neurotropic virus, and the immune system's hyperactivation has a vital role in its complications (table 8). We also reported the drug candidates that were categorized as inflammatory drugs or antiviral drugs. We have reported forty-four drugs with immunomodulatory or immunosuppressive functions that have validated effects in treating inflammatory or autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Chron's disease, ulcerative colitis, systemic lupus erythematosus, and Behcet's disease (Suzuki Kurokawa and Suzuki 2004, Allison 2005, Potthast, Dressman et al. 2005, Klotz, Teml et al. 2007, Mease 2007, Lucas 2016, Corbett, Chehadah et al. 2017, Laurence L. Brunton 2018, Yang, Wu et al. 2018, Afra, Razmi et al. 2019). Ten antiviral medications (mostly anti-HIV-1 and hepatitis) were also among our repurposed drugs. For example, Hydroxychloroquine and chloroquine were two drugs reported to inhibit the viral entry by changing the endosomal PH and disrupting glycosylation of ACE2 (Vincent, Bergeron et al. 2005, Plantone and Koudriavtseva 2018, Devaux, Rolain et al. 2020, Wang, Cao et al. 2020).
Our repurposed medications that have validated effects in the nervous system included 1-Risperidone (schizophrenia and bipolar disorders), 2-Ibudilast (neuroprotective in multiple sclerosis), 3-Carbamazepine (control seizures, trigeminal neuralgia, and Bipolar disease), 4-Midazolam (hypnotic-sedative, muscle relaxant and anticonvulsant ), 5-pyridoxine (synthesis of the neurotransmitters), 6-Duloxetine (neuropathic pain and Generalized Anxiety disorder), 7-melatonin (sleep-wake cycle disturbances), 8-Rizatriptan (migraine), and 9-selective serotonin receptor inhibitors (antidepressant) (Tambasco-Studart, Titiz et al. 2005, Chen and Lin 2012, Tolou-Ghamari, Zare et al. 2013, Kennedy, Lam et al. 2016). We have also discussed Ibudilast's neuroprotective properties, Melatonin, Pyridoxine, and SSRIs, to validate the repurposed drugs.
Ibudilast suppresses the excess production of pro-inflammatory cytokines such as interleukin IL-1β, IL-6, and TNF-α in the CNS. This anti-inflammatory property can help treat other viral-related neurocognitive disorders, such as HIV-associated neurocognitive disorders (HAND) and other neuro-inflammatory diseases [155-157]. Our SARS-CoV-2 treated cells in-silico results also showed the up-regulation of IL-1β, IL-6, and TNF-α. Therefore, it can be recommended as a possibly suitable repurposed drug for COVID-19 neural manifestations' investigations (in vitro and in vivo).
Melatonin seems a suitable candidate in treating COVID-19 since it shows excellent anti-oxidative properties by directly scavenging free radicals and stimulating antioxidant enzymes. Furthermore, Melatonin has anti-inflammatory effects by reducing pro-inflammatory cytokines and balancing innate immune response's over-activation while promoting adaptive immunity [158, 159]. The published reports related to Melatonin used in the animals with deadly viral infections such as Venezuelan equine encephalomyelitis virus (VEEV), Semliki Forest virus (SFV), and West Nile virus (WNV) showed that Melatonin is not viricidal. However, somewhat it reduces the severity of viral infections [158, 159]. Adequate Pyridoxine supplementation can prevent polyneuropathy, which is the most common neurological complication associated with HIV [160, 161]. Perhaps, Pyridoxine can be helpful in such kinds of pains in COVID-19 patients.
Serotonin shows immunomodulatory properties by downregulating central and peripheral inflammatory responses. So selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (with a clinical trial ID of NCT04377308) and Fluvoxamine (NCT04342663) could effectively dampen the excessive production of cytokines and prevent neurological complications in various neurocognitive disorders and COVID-19 [162].
We investigated the number of shared medicines between our list and the list of all registered drugs for COVID-19 clinical trials to verify our repurposed medications for their possible use in treating neural manifestations of COVID-19. Fifty-four of our repurposed medications were previously registered for investigations against COVID-19 (table 8).