PMC8695435_01
Graphic abstract. 304 COVID-19 in-hospital patients were enrolled in this study to screen out those with prominent new-onset neurological manifestations. Finally, 8 COVID-19 patients with neurological manifestations and three non-COVID-19 patients were selected. NGS and proteomics analysis were conducted in the simultaneously obtained CSF and serum samples from the selected patients. SARS-CoV-2 RNA were validated to present in the CSF of COVID-19 patients, providing the direct clue that SARS-CoV-2 may invade central nervous system. The proteomic results highlighted the involvement of complement system, PI3K-Akt signaling pathway, enhanced cellular interaction, and macrophages in pathogenesis of the COVID-19 related neurological manifestations.
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Figure 1 ). For the eight patients, the median age was 65.5 years old and two of them were female. Tomography (CT) ( Figure S1 ). The period from admission to the onset of neurological symptoms/signs ranged from 1 to 33 days ( Figure 2A ).
The timeline of clinical events engaged in eight COVID-19 patients and the consecutive routine tests. (A), The illustration summaries the hospitalization duration, the onset time of neurologic symptoms/signs, the time-points of lumbar puncture, and the outcomes of 8 COVID-19 patients with neurological manifestations. (B), Continuous routine tests about hemogram, enzymonram and coagulation for 8 COVID-19 patients. The semitransparent yellow rectangles represent the normal ranges for different parameters. The horizontal ordinates all indicate the times of tests after admission.
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Table 1 ). Their median hospitalization duration was 40.5 days (IQR, 35-53) ( Table 1  and  Figure 2A ). Figure 2B ). For these eight patients, two recovered and were discharged while the other six patients eventually died from respiratory and circulatory failure, multiple organ failure, or septic shock ( Table 1 ). Tomography (CT) ( Figure S1 ). The period from admission to the onset of neurological symptoms/signs ranged from 1 to 33 days ( Figure 2A ). The Gene Oncology analysis also yielded less results with statistically significance ( Figure S2 ).
Visualization of SARS-CoV-2 RNA-Seq Read. For each CSF sample, total RNA was extracted and used for RNA-seq library preparation after the rRNA removal. Then the cDNA libraries were applied to Illunima Novaseq 6000 system for 150 nt paired-end sequencing. Reads unique to SARS-CoV-2 were mapped on the SARS-CoV-2 genome. The chart illustrates the distribution of SARS-CoV-2-specific reads on SARS-CoV-2 genome from the eight CSF samples in histograms (red) and read clusters (blue).
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SARS-CoV-2 RNA sequences were indeed detected unequivocally in the CSF samples from four patients, and CSF samples of the four remaining patients were deemed likely to contain SARS-CoV-2 RNA sequences ( Figure 3 ).
DEPs of CSF from 8 COVID-19 patients with Neurologic Manifestations vs Control patients. (A), Volcano plot for identified proteins in CSF between 8 COVID-19 patients and 3 control patients. Proteins with fold-change beyond 1.2 or below -1.2 with p value lower than 0.05 were considered as significantly differential expression. (B, C) KEGG Enrichment Pathway and GO Oncology analysis of differentially expressed proteins with significance (n =185) that corresponded in the (A). (D) The diagram shows 185 proteins in CSF and 59 in serum were significantly altered in COVID-19 vs control groups. Note that only 20 proteins were in common, indicating the limited overlap. Venn diagrams showing that there were 20 proteins shared between CSF DEPs and Serum DEPs. 19 shared proteins were consistently downregulated in both samples while one protein (marked with asterisk in A) showed the opposite change in two samples.
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Among the DEPs in the CSF, the prominent marker C-reactive protein (CRP) was increased with the highest fold change ( Figure 4A ), indicating an active inflammation in the CNS. Among these differentially expressed proteins (DEPs) from CSF vs serum samples, only 20 proteins were in common and were all down-regulated except ECM1 showed upregulation in CSF ( Figure 4D ). The analysis revealed that the highest proportion of DEPs (14 proteins or 13.7%) were those involved in complement and coagulation cascades ( Figure 4B ), including membrane cofactor protein (MCP; CD46), membrane attack complex inhibitory protein (CD59), complement C1q-like protein 3 (C1QL3), complement C4 binding protein B (C4BPB), carboxypeptidase B2 (CPB2), complement C3, C6, and C8a ( Figures 4A  and  6 ). The PI3K-Akt signaling pathway was the second most affected pathway, with 12 DEPs identified by the KEGG pathway analysis ( Figures 4B  and  6 ). Figure 4C ). For further comprehensive understanding of resuts, manual inspection of other DEPs in reference to the UniProt database allowed for assigning them into several functional clusters, belonging to pathways such as neuronal growth and signaling, and cell adhesion ( Figures 6B, C ).
The correlation between the original protein expression profile of CSF and serum samples in 8 COVID-19 patients and Volcano Plots relevant to different subgroup comparisons. (A) Venn diagrams showing the number of all proteins identified and quantified by proteomics in CSF and serum of 8 COVID-19 patients and their intersections with DEPs generated in different comparison patterns. (B) Heatmaps of expression levels of 313 proteins shared by CSF and serum proteomic profile. (C) Volcano plot for identified proteins in CSF between CSF SARS-CoV-2 positive (4) and negative (4) patients. (D) Volcano plot for identified proteins in CSF between deceased (6) and survived (2) patients. Proteins with fold-change beyond 1.2 or below -1.2 with p value lower than 0.05 were considered as significantly differential expression.
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This contrasting protein abundance may reflect more active perturbation occurred in central nervous system and were reminiscent of the prominent neurological manifestations in these patients. 313 proteins were shared by these two samples, which accounted for 20.1% (313/1491) of total proteins in CSF ( Figure 5A ). As illustrated by heatmap, the expression level of these shared proteins also presented great polarity in the two sets of samples ( Figure 5B ). As expected, there exited less DEPs ( Figures 5C, D ) and less KEGG enrichment pathway engaged. As mentioned above, the marker CRP also present highest fold change in the CSF between the 6 deceased and 2 survived patients, which indicated its potential to predict the prognosis ( Figure 5D ).
Heatmap of 185 DEPs in CSF and expression values of selected DEPs. (A) Heatmap of significant DEPs (n=185) in CSF of eight COVID-19 patients vs three non-COVID-19 control patients. (B) Heatmap of selected DEPs enriched in KEGG pathway analysis or found to be important after manual inspection. (C) The expression level (Original value) of representative DEPs in CSF between COVID-19 and control cases. o; *P<0.05, and **P<0.001 by two-tailed unpaired Student t test.
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The analysis revealed that the highest proportion of DEPs (14 proteins or 13.7%) were those involved in complement and coagulation cascades ( Figure 4B ), including membrane cofactor protein (MCP; CD46), membrane attack complex inhibitory protein (CD59), complement C1q-like protein 3 (C1QL3), complement C4 binding protein B (C4BPB), carboxypeptidase B2 (CPB2), complement C3, C6, and C8a ( Figures 4A  and  6 ). The PI3K-Akt signaling pathway was the second most affected pathway, with 12 DEPs identified by the KEGG pathway analysis ( Figures 4B  and  6 ). Figure 4C ). For further comprehensive understanding of resuts, manual inspection of other DEPs in reference to the UniProt database allowed for assigning them into several functional clusters, belonging to pathways such as neuronal growth and signaling, and cell adhesion ( Figures 6B, C ).