Hypothesis: Long COVID brain fog is caused by free glycan sugar chains in the brain

by Angela May O’Connor, Ph.D. ¹

¹Independent Patient-Researcher

Cite as: O’Connor, A. M. (2023). Hypothesis: Long COVID brain fog is caused by free glycan sugar chains in the brain. Patient-Generated Hypotheses Journal for Long COVID & Associated Conditions, Vol. 1, 5-12

Abstract

Coronavirus spike proteins are composed of glycoproteins, molecules that have a protein center and sugar side-chains. These coronavirus glycans interact with other protein-sugar molecules present on the outer surface of cells, known as the glycocalyx. Coronavirus spike proteins particularly interact with the glycocalyx within blood vessels, causing these protein-sugars to peel off the internal walls of blood vessels. Previous work has shown that during sepsis, protein-sugar molecules similarly slough off the internal walls of blood vessels, cross the blood-brain barrier and contribute to cognitive, memory, and mood disorders. This proposal hypothesizes that viral persistence and constant coronavirus spike protein presence in the bloodstream of Long COVID patients similarly causes an ongoing degradation of endothelial glycocalyx, resulting in free floating glycan sugar side-chains that contribute to the cognitive issues observed in this condition.

Introduction

Proteoglycans are a broad group of molecules, comprised of a core protein surrounded by sugar side-chains that are composed of differentially sulphated groups of glycosaminoglycans (GAG); both the core protein and sugar chains determine binding affinity and functionality of a proteoglycan^1,2. The spike proteins found on the external surface of SARS-CoV-2, the virus that causes COVID-19, are glycoproteins, also composed of a protein core and sugar side-chains^3-5. During normal function, sugar side-chains are constrained to their core proteins and do not interfere with normal biological processes; however, free glycan side-chains can damage various tissues, such as during sepsis^6-8.

These glycan fragments are small enough to pass the blood-brain barrier, resulting in impaired memory and cognitive functions both during and after sepsis, thought to be due to the presence of free glycan side-chains within the brain^6,7. The SARS-CoV-2 proteoglycan spike protein is known to persistently circulate in patients with Long COVID¹⁰, and is known to both cross and disrupt the function of the blood-brain barrier^11-13. This proposal addresses the possibility that freely circulating, persistent SARS-CoV-2 spike protein is a major contributor to the cognitive and memory deficits experienced by patients with Long COVID.

Hypothesis

Proteoglycans play many different roles in cellular and biological processes, particularly heparan sulfate proteoglycans (HSPGs)² like those thought to act as a co-receptor for the SARS-CoV-2 virus^3-5. HSPGs mediate growth factor signaling, provide guidance during cellular migration and axonal growth within the brain, organize the extracellular matrix, enable cellular motility and adhesion, and facilitate virus-host interactions among other cell-cell crosstalk². HSPGs may be secreted into and participate in the extracellular matrix, form intracellular secretory vesicles, or be bound to cellular membranes², where they act as co-receptors for various growth factors, proteins and the SARS-CoV-2 virus^3-5.

Proteoglycans and glycoproteins within the brain regulate neuronal health, form chemical gradients that guide cellular migration during development, provide cellular support, enable synaptic connections, regulate the availability and signaling of growth factors, and close off critical periods by helping to solidify mature neural networks^{2, 14-18}. It has been seen that altering proteoglycans within the rodent brain results in altered behavior^19-21 and neural function^22,23.

Recent work has established the presence of free-floating SARS-CoV-2 spike protein within the blood of Long COVID patients⁸. It is hypothesized that viral persistence within patients results in a variety of immunological, neurological, and autonomic dysregulation, while also providing a constant supply of the spike glycoprotein. The presence of freely circulating glycans has also been detected, and significantly contributes to pathophysiology during sepsis^6,7. Like Long COVID, sepsis is a multi-organ, multi-system inflammatory process where normal cellular activity is disrupted or completely breaks down, and the two have been compared as having similar long-term sequelae³⁵. Proteoglycans and glycoproteins that are sloughed off blood vessel endothelia during sepsis are capable of crossing the blood-brain barrier, and are thought to contribute to the ongoing cognitive and neurological issues seen in post-sepsis syndrome^6-9. Similarly, the free floating spike protein found in the blood of Long COVID patients is also capable of crossing the blood-brain barrier^12,13, and high levels of HSPGs have been found in the blood of COVID-19 patients due to endothelial glycocalyx disruption³. What is not known is whether the presence of these glycans in the brain contributes to the cognitive and neurological issues seen in Long COVID, similar to the effect observed in septic and post-septic patients.

This proposal puts forward the hypothesis that the circulating SARS-CoV-2 spike protein found in Long COVID patients crosses the blood-brain barrier and interferes with normal neuronal and cellular functioning within the brain, contributing to the brain fog, speech impediments, memory issues, cognitive impairments, and other neurological disorders observed in patients with Long COVID.

How to test the hypothesis

Assessing patient tissue

Assess proteoglycan and glycoprotein sugar side-chain recovery from post-mortem tissue taken from the brains of severe acute COVID-19 patients. Recover glycan chains and perform liquid chromatography mass spectrometry using established techniques⁷ to assess the presence of the SARS-CoV-2 spike protein or free-floating HSPG sugar side-chains within various brain regions.
Correlate circulating glycan levels with brain glycan measurements and behavioral/cognitive performance to determine how free floating SARS-CoV-2 spike protein and elevated HSPG blood levels correlate with impaired cognitive capabilities.

Assessing animal studies

Use an established Long COVID animal model such as that published by Frere et al. (2022)³⁰. Compare behavioral performance in the Puzzle-Box to assess cognitive capabilities²⁹ across a Long COVID animal model, sepsis animal model, and possibly animal model with disturbed brain glycans (i.e. chondroitinase and heparinase injections into the hippocampus).
Fresh frozen brains from the above animals: recover glycan chains and perform liquid chromatography mass spectrometry⁷ to assess the presence of the SARS-CoV-2 spike protein and free-floating HSPG sugar side-chains within various brain regions. Stain brain samples for 3G10 and 10EF to assess the presence of broken/free floating proteoglycan side-chains versus bound/complete side-chains²⁷, and for perineuronal nets using wisteria floribunda agglutinin (WFA) to assess the state of the glycans within the brain extracellular matrix²⁸.
Collect trunk blood at sac: process for the presence of free floating proteoglycans and glycoproteins, including the SARS-CoV-2 spike protein.
Correlate circulating glycan levels with brain glycan measurements and behavioral performance to determine how much free floating glycans, including the SARS-CoV-2 spike protein, contribute to impaired cognitive capabilities.

Potential therapeutics

Nattokinase to dissolve persistent circulating SARS-CoV-2 spike protein³¹
Synthetic heparan sulfate mimetics to disrupt glycocalyx degradation caused by persistent SARS-CoV-2 spike protein³²
Drugs used to restore endothelial functioning in diabetes treatment^33,34

Unanswered questions

1. Do Long COVID patients have ongoing endothelial glycocalyx degradation similar to that observed in acute COVID patients?

2. What neurological or cognitive impacts do the free-floating SARS-CoV-2 spike protein glycans have in patients with Long COVID?

3. How can we measure or determine this?

4. What are some ways in which we can eliminate the free floating glycans from Long COVID patients’ tissues, particularly within the brain?

References

¹ Avram, S., Shaposhnikov, S., Buiu, C., & Mernea, M. (2014). Chondroitin sulfate proteoglycans: Structure-function relationship with implication in neural development and brain disorders. BioMed Research International, 2014, 1–11. https://doi.org/10.1155/2014/642798

² Sarrazin, S., Lamanna, W. C., & Esko, J. D. (2011). Heparan sulfate proteoglycans. Cold Spring Harbor Perspectives in Biology, 3(7), a004952. https://doi.org/10.1101/cshperspect.a004952

³ Choi, Y., Cao, Y., Frank, M., Woo, H., Park, S. M., Yeom, M. S., Croll, T. I., Seok, C., & Im, W. (2021). Structure, dynamics, receptor binding, and antibody binding of the fully glycosylated full-length SARS-CoV-2 spike protein in a viral membrane. Journal of Chemical Theory and Computation, 17(4), 2479–2487. https://doi.org/10.1021/acs.jctc.0c01144

⁴ Zhang, J., Xiao, T., Cai, Y., & Chen, B. (2021). Structure of SARS-CoV-2 spike protein. Current Opinion in Virology, 50, 173–182. https://doi.org/10.1016/j.coviro.2021.08.010

⁵ De Pasquale, V., Quiccione, M. S., Tafuri, S., Avallone, L., & Pavone, L. M. (2021). Heparan sulfate proteoglycans in viral infection and treatment: A special focus on SARS-CoV-2. International Journal of Molecular Sciences, 22(12), 6574. https://doi.org/10.3390/ijms22126574

⁶ Sullivan, R. J., Rockstrom, M. D., Schmidt, E. W., & Hippensteel, J. A. (2021). Endothelial glycocalyx degradation during sepsis: Causes and consequences. Matrix Biology Plus, 12, 100094. https://doi.org/10.1016/j.mbplus.2021.100094

⁷ Zhang, X., Han, X., Xia, K., Xu, Y., Yang, Y., Oshima, K., Haeger, S. M., Perez, M. M., McMurtry, S. A., Hippensteel, J. A., Ford, J. A., Herson, P. S., Liu, J., Schmidt, E. W., & Linhardt, R. J. (2019). Circulating heparin oligosaccharides rapidly target the hippocampus in sepsis, potentially impacting cognitive functions. Proceedings of the National Academy of Sciences of the United States of America, 116(19), 9208–9213. https://doi.org/10.1073/pnas.1902227116

⁸ Chelazzi, C., Villa, G., Mancinelli, P., De Gaudio, A. R., & Adembri, C. (2015). Glycocalyx and sepsis-induced alterations in vascular permeability. Critical Care, 19(1). https://doi.org/10.1186/s13054-015-0741-z

⁹ Logsdon, A. F., Rhea, E. M., Reed, M. J., Banks, W. A., & Erickson, M. A. (2021). The neurovascular extracellular matrix in health and disease. Experimental Biology and Medicine, 246(7), 835–844. https://doi.org/10.1177/1535370220977195

¹⁰ Swank, Z., Senussi, Y., Manickas-Hill, Z., Yu, X. G., Li, J., Alter, G., & Walt, D. R. (2022). Persistent circulating severe acute respiratory syndrome coronavirus 2 spike Is associated with post-acute coronavirus disease 2019 sequelae. Clinical Infectious Diseases. https://doi.org/10.1093/cid/ciac722

¹¹ DeOre, B. J., Tran, K. A., Andrews, A. M., Ramirez, S. H., & Galie, P. A. (2021). SARS-CoV-2 spike protein disrupts blood–brain barrier Integrity via RhoA activation. Journal of Neuroimmune Pharmacology, 16(4), 722–728. https://doi.org/10.1007/s11481-021-10029-0

¹² Petrovszki, D., Walter, F. R., Vigh, J. P., Kocsis, A., Valkai, S., Deli, M. A., & Dér, A. (2022). Penetration of the SARS-CoV-2 spike protein across the blood–brain barrier, as revealed by a combination of a human cell culture model system and optical biosensing. Biomedicines, 10(1), 188. https://doi.org/10.3390/biomedicines10010188

¹³ Rhea, E. M., Logsdon, A. F., Hansen, K. T., Williams, L. W., Reed, M. J., Baumann, K. K., Holden, S., Raber, J., Banks, W. A., & Erickson, M. A. (2021). The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice. Nature Neuroscience, 24(3), 368–378. https://doi.org/10.1038/s41593-020-00771-8

¹⁴ Celio, M. R., & Blümcke, I. (1994). Perineuronal nets — A specialized form of extracellular matrix in the adult nervous system. Brain Research Reviews, 19(1), 128–145. https://doi.org/10.1016/0165-0173(94)90006-x

¹⁵ Celio, M. R., Spreafico, R., De Biasi, S., & Vitellaro-Zuccarello, L. (1998). Perineuronal nets: Past and present. Trends in Neurosciences, 21(12), 510–515. https://doi.org/10.1016/s0166-2236(98)01298-3

¹⁶ Sorg, B. A., Berretta, S., Blacktop, J. M., Fawcett, J. W., Kitagawa, H., Kwok, J. C. F., & Miquel, M. (2016). Casting a wide net: Role of perineuronal nets in neural plasticity. The Journal of Neuroscience, 36(45), 11459–11468. https://doi.org/10.1523/jneurosci.2351-16.2016

¹⁷ Condomitti, G., Wierda, K. D., Schroeder, A., Rubio, S. E., Vennekens, K. M., Orlandi, C., Martemyanov, K. A., Gounko, N. V., Savas, J. N., & De Wit, J. (2018). An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 Synapses. Neuron, 100(1), 201-215.e9. https://doi.org/10.1016/j.neuron.2018.08.038

¹⁸ Kamimura, K., Odajima, A., Ikegawa, Y., Maru, C., & Maeda, N. (2019). The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway. Cell Reports, 28(12), 3144-3156.e4. https://doi.org/10.1016/j.celrep.2019.08.032

¹⁹ Hylin, M. J., Orsi, S. A., Moore, A. T., & Dash, P. K. (2013). Disruption of the perineuronal net in the hippocampus or medial prefrontal cortex impairs fear conditioning. Learning & Memory, 20(5), 267–273. https://doi.org/10.1101/lm.030197.112

²⁰ Lee, H., Leamey, C. A., & Sawatari, A. (2012). Perineuronal nets play a role in regulating striatal function in the mouse. PLOS ONE, 7(3), e32747. https://doi.org/10.1371/journal.pone.0032747

²¹Karlsson-Lindahl, L., Schmidt, L., Haage, D., Hansson, C., Taube, M., Egeciouglu, E., Tan, Y., Admyre, T., Jansson, J., Vlodavsky, I., Li, J., Lindahl, U., & Dickson, S. L. (2012). Heparanase affects food intake and regulates energy balance in mice. PLOS ONE, 7(3), e34313. https://doi.org/10.1371/journal.pone.0034313

²²Hayani, H., Song, I., & Dityatev, A. (2018). Increased excitability and reduced excitatory synaptic input into fast-spiking CA2 interneurons after enzymatic attenuation of extracellular matrix. Frontiers in Cellular Neuroscience, 12. https://doi.org/10.3389/fncel.2018.00149

²³ Yamada, J., Ohgomori, T., & Jinno, S. (2015). Perineuronal nets affect parvalbumin expression in GABAergic neurons of the mouse hippocampus. European Journal of Neuroscience, 41(3), 368–378. https://doi.org/10.1111/ejn.12792

²⁴ Grobbelaar, L. M., Venter, C., Vlok, M., Ngoepe, M., Laubscher, G. J., Lourens, P. J., Kell, D. B., & Kell, D. B. (2021). SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19. Bioscience Reports, 41(8). https://doi.org/10.1042/bsr20210611

²⁵ Pretorius, E., Venter, C., Laubscher, G. J., Kotze, M. J., Oladejo, S., Watson, L. R., Rajaratnam, K., Watson, B. W., & Kell, D. B. (2022). Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-acute sequelae of COVID-19 (PASC). Cardiovascular Diabetology, 21(1). https://doi.org/10.1186/s12933-022-01579-5

²⁶ Kell, D. B., Laubscher, G. J., & Pretorius, E. (2022). A central role for amyloid fibrin microclots in Long COVID/PASC: Origins and therapeutic implications. Biochemical Journal, 479(4), 537–559. https://doi.org/10.1042/bcj20220016

²⁷ Fuxe, K., Chadi, G., Tinner, B., Agnati, L. F., Pettersson, R. F., & David, G. (1994). On the regional distribution of heparan sulfate proteoglycan immunoreactivity in the rat brain. Brain Research, 636(1), 131–138. https://doi.org/10.1016/0006-8993(94)90187-2

²⁸ Costa, C. M., Tortosa, R., Domènech, A., Vidal, E., Pumarola, M., & Bassols, A. (2007). Mapping of aggrecan, hyaluronic acid, heparan sulphate proteoglycans and aquaporin 4 in the central nervous system of the mouse. Journal of Chemical Neuroanatomy, 33(3), 111–123. https://doi.org/10.1016/j.jchemneu.2007.01.006

²⁹ O’Connor, A., Burton, T. M., Leamey, C. A., & Sawatari, A. (2014). The use of the puzzle box as a means of assessing the efficacy of environmental enrichment. Journal of Visualized Experiments, 94. https://doi.org/10.3791/52225

³⁰ Frere, J. J., Serafini, R. A., Pryce, K. D., Zazhytska, M., Oishi, K., Golynker, I., Panis, M., Zimering, J., Horiuchi, S., Hoagland, D. A., Møller, R., Ruiz, A., Kodra, A., Overdevest, J. B., Canoll, P., Borczuk, A. C., Chandar, V., Bram, Y., Schwartz, R. S., Lomvardas, S., Zachariou, V., & tenOever, B. R. (2022). SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery. Science Translational Medicine, 14(664). https://doi.org/10.1126/scitranslmed.abq3059

³¹ Tanikawa, T., Kiba, Y., Yu, J. B., Hsu, K., Chen, S., Ishii, A., Yokogawa, T., Suzuki, R., Inoue, Y., & Kitamura, M. (2022). Degradative effect of nattokinase on spike protein of SARS-CoV-2. Molecules, 27(17), 5405. https://doi.org/10.3390/molecules27175405

³² Guimond, S. E., Mycroft-West, C. J., Gandhi, N. S., Tree, J. A., Le, T., Spalluto, C., Humbert, M. V., Buttigieg, K. R., Coombes, N., Elmore, M. J., Wand, M. E., Nyström, K., Said, J., Setoh, Y. X., Amarilla, A. A., Modhiran, N., Sng, J. D. J., Chhabra, M., Young, P. M., . . . Turnbull, J. E. (2022). Synthetic heparan sulfate mimetic pixatimod (PG545) potently inhibits SARS-CoV-2 by disrupting the spike–ACE2 interaction. ACS Central Science, 8(5), 527–545. https://doi.org/10.1021/acscentsci.1c01293

³³ Stirban, A., Negrean, M., Stratmann, B., Gawlowski, T., Horstmann, T., Götting, C., Kleesiek, K., Mueller-Roesel, M., Koschinsky, T., Uribarri, J., Vlassara, H., & Tschoepe, D. (2006). Benfotiamine prevents macro- and microvascular endothelial dysfunction and oxidative stress following a meal rich in advanced glycation end products in individuals with type 2 diabetes. Diabetes Care, 29(9), 2064–2071. https://doi.org/10.2337/dc06-0531

³⁴ Qiu, Y., Buffonge, S., Ramnath, R. D., Jenner, S., Barton, S., Arkill, K. P., Neal, C. J., Verkade, P., White, S., Hezzell, M. J., Salmon, A., Suleiman, M., Welsh, G. I., Foster, R. J., Madeddu, P., & Satchell, S. C. (2022). Endothelial glycocalyx is damaged in diabetic cardiomyopathy: Angiopoietin 1 restores glycocalyx and improves diastolic function in mice. Diabetologia, 65(5), 879–894. https://doi.org/10.1007/s00125-022-05650-4

³⁵ Sepsis Alliance. (2022, June 29). Post-Sepsis Syndrome: Sepsis Alliance. https://www.sepsis.org/sepsis-basics/post-sepsis-syndrome/