Review 2: "SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-1"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

The preprint titled "SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in Post COVID-19," authored by Rong et al., explores the presence of the spike protein of SARS-CoV-2 in the brain tissue of mouse models and post-mortem human samples. The objective is to investigate its potential long-term neurological effects. Optical tissue clearing was utilized to identify tissues where the spike protein accumulated in mice, and the distribution of the spike protein in post-mortem samples from COVID-19 patients was examined. The study's findings indicate a significant accumulation of the spike protein in various areas, including the skull marrow, brain meninges, and brain parenchyma. This suggests the possibility of the spike protein traversing the skull-meninges-brain axis and potentially affecting long-term neurological function.

High-resolution mass spectrometry-based proteomics was employed to identify proteins with altered expression in the brain region due to the presence of the spike protein. This approach helps identify proteins that may be impacted by the spike protein and provides insights into the molecular mechanisms underlying its potential neurological effects. The study identified disregulation in complement and coagulation cascades, neutrophil-related pathways, and an upregulation of pro-inflammatory proteins. The discovery of spike protein accumulation in the skull marrow, brain meninges, and brain parenchyma is significant as it sheds light on the potential mechanisms through which SARS-CoV-2 may affect the central nervous system. Further research is needed to investigate the long-term neurological implications of COVID-19 and the development of post-COVID-19 neurological complications.

Although the study's main claims are generally supported by the methods and data, there are areas that could be improved to enhance the manuscript's quality. The study primarily focuses on the accumulation of the spike protein within the skull-meninges-brain axis, providing valuable insights but not encompassing the entire central nervous system. The potential effects of the spike protein on other brain regions or neural networks beyond the skull-meninges-brain axis are not extensively explored. Additionally, the research relies on mouse models and post-mortem human tissues, which may not fully represent the dynamic nature of the living human brain during active infection or post-recovery. Furthermore, the study concentrates on the spike protein's accumulation itself rather than investigating the broader interactions between the virus and the brain. A comprehensive understanding of the potential neurological complications associated with COVID-19 would involve exploring the mechanisms of viral entry into the central nervous system and its subsequent effects on neural cells and processes.

To delve deeper into the functional roles of the key proteins identified in altered signaling pathways due to spike proteins, additional techniques like immunohistochemistry or Western blot can validate their significance within the cellular context. Further studies, including in-vivo experiments and longitudinal analyses, are necessary to validate and expand upon the findings presented in this preprint, as well as address these limitations. A multidisciplinary approach incorporating virology, neurology, and immunology would provide a more comprehensive understanding of the long-term neurological consequences of SARS-CoV-2 infection.