The ongoing pandemic of COVID-19 is caused by the betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and causes a spectrum of disease from asymptomatic to critical. The rapid transmission of the virus is responsible for the difficulty in containing it, coupled with the significant percentage of asymptomatic transmission.
A recent study published in the journal Pain in October 2020 adds another piece to the puzzle, showing that the virus is capable of dulling pain pathways. This may contribute to its early and extensive spread.
Asymptomatic Patients and the Spread of COVID-19
Asymptomatic and minimally symptomatic patients make up ~40% of all SARS-CoV-2 cases, according to recent data released by the US Centers for Disease Control and Prevention, and about half of all transmission occurs before the onset of the earliest symptom. These individuals are, therefore, efficient spreaders of the virus, which has led to huge challenges in preventing viral transmission. On the other hand, symptomatic patients often suffer from pain, as in headache, myalgia, arthralgia, and abdominal discomfort.
The question raised here is, do asymptomatic or minimally symptomatic patients also have the same viral processes or are pain pathways silenced somehow?
The virus enters and infects the host cell after binding to the angiotensin-converting enzyme 2 (ACE2), the receptor for the virus on the host cell. However, there is a smaller group of neurons on which the ACE2 receptor is expressed, which connects to the spinal column and the brainstem neurons. These are thought to be responsible for headaches and neuralgic pain—however, few neurons in total express ACE2.
Alternatives to ACE2
The ACE2 levels are lower with advancing age, though this correlates inversely with the severity of the disease. This may suggest that there are other receptors than ACE2 to mediate viral-cell binding.
In fact, two reports have recently shown that the spike protein of the virus binds to a receptor called the neuropilin-1 receptor (NRP-1), at the b1b2 domain. The region responsible for this binding is a polybasic sequence not found in either SARS or MERS viruses, and it is called the C-end rule. This interaction promotes viral entry into the cell.
Analysis of the proteins, transcribed RNA fragments and other cellular processes show that the level of NRP-1 is higher in samples from patients with COVID-19 than in healthy individuals.
Importance of VEGF-A
Under physiological conditions, the b1b2 domain is bound by a factor called VEGF-A (vascular endothelial growth factor-A), which is important in inflammation as well as in wound healing. The researchers, therefore, explored whether the presence of the spike protein of SARS-CoV-2 is inhibitory to the binding of this ligand with its receptor, and thus whether it affects the signaling through pain pathways.
In both rodents and humans, VEGF-A is known to promote the sensation of pain. This factor is found at elevated levels in the bronchial alveolar lavage fluid obtained from COVID-19 patients, but there is a significantly lower level in the serum of asymptomatic patients compared to symptomatic individuals. The researchers, therefore, also explored whether the spike protein could also cause the pain sensation to recede.
Binding to NRP-1 Induces Pain
The researchers found that when the NRP-1 receptor is bound by its ligand VEGF-A, the spontaneous firing rate of the dorsal root ganglion (DRG) neurons increased. This was blocked by the S1 domain of the spike protein and by the NRP-1 inhibitor EG00229.
Other ligands for receptors that act as co-receptors for NRP-1 failed to potentiate the firing of these nociceptive cells. Thus, this indicates a new pathway for pain, namely, the VEGF-A ligand and the NRP-1 receptor.
The viral spike protein binds to the same binding pocket on NRP-1, preventing pain signaling via this pathway. While VEGF-A was confirmed to potentiate the sensation of pain following both mechanical and thermal stimuli, these actions were blocked by either the binding of the NRP-1 receptor by the spike protein or the NRP-1 inhibitor EG00229. However, neither of these is effective against pain by itself.
Spike Protein Inhibits VEGF-A-Mediated Increase in DRG Ion Currents
The researchers found that the binding of VEGF-A doubled the total sodium and calcium ion currents in the DRG neurons, but this was also blocked by the spike protein as well as the NRP-1 inhibitor EG00229. However, these ligands alone did not cause any change in sodium or calcium currents.
VEGF-A Enhances Synaptic Activity in Dorsal Horn
The synaptic activity within the lumbar dorsal horn was found to be increased in the presence of VEGF-A, not in terms of the amplitude of the excitatory post-synaptic current but its frequency which underwent an almost fourfold increase. The magnitude of this increase was reduced by 50% and 57% by the spike protein and EG00229. This suggests that these molecules have a presynaptic action, and antagonize pain signaling in this pathway.
In a rat model, the administration of either spike protein or EG00229 was capable of completely inhibiting VEGF-A signaling in chronic neuropathic pain, thus increasing the pain threshold following injury.
In fact, according to researcher Rajesh Khanna, "The spike protein completely reversed the VEGF-induced pain signaling. It didn't matter if we used very high doses of spike or extremely low doses."
Implications
The study shows that the activity of VEGF-A, a chemical that sensitizes the pain pathway and gives rise to pain, is inhibited by the spike protein as well as the NRP-1 inhibitor EG00229. The experimental results demonstrate that the SARS-CoV-2 spike protein efficiently inhibits pain signaling in the VEGF-A/NRP-1 pathway. In chronic neuropathic pain, spike protein blocked the VEGF-A-induced increases in both sodium and calcium current densities, reduced spontaneous firing in DRG neurons, synaptic transmission in the lumbar dorsal spinal neurons, and pain due to mechanical or thermal stimulation following injury.
In COVID-19 patients, the observed high levels of VEGF-A would be expected to be associated with increased pain via the NRP-1 pathway. These results suggest that the spike protein usurps the binding domain of NRP-1 typically occupied by VEGF-A, thus reducing the pain sensed by this pathway.
Said Michael D. Dake, University of Arizona Health Sciences Senior Vice President, "This research raises the possibility that pain, as an early symptom of COVID-19, may be reduced by the SARS-CoV-2 spike protein as it silences the body's pain signaling pathways."
Khanna explains, "Perhaps the reason for the unrelenting spread of COVID-19 is that in the early stages, you're walking around all fine because your pain has been suppressed." The researchers plan to continue work on neuropilin as a mechanism of the early spread of COVID-19.
Secondly, it is conceivable that the spike protein could serve as the basis for a family of novel pain-alleviating molecules that act through NRP-1 inhibition. However, alternative fragments of the spike protein or other viral proteins may promote pain, and this needs to be explored in future studies. The sequence of molecular events that occur following the VEGF-A/NRP-1 binding also remains to be elucidated.
The NRP-1 antibody MNRP1685A is undergoing clinical trials. The latter prevents NRP-1 binding by VEGF-A, but associated neuropathy has been observed. This could be due to the inhibition of the alternative spliced isoform VEGF-A165b, which is neuroprotective rather than an analgesic.
The current study thus provides a rationale for examining the effect of targeted inhibition of the VEGF-A/NRP-1 pro-nociceptive signaling axis rather than a broad inhibition of VEGF-A as with cancer therapeutic monoclonal antibody bevacizumab.
Khanna says, "We are moving forward with designing small molecules against neuropilin, particularly natural compounds, that could be important for pain relief.'
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