Increased Cerebrospinal Fluid Angiotensin-Converting Enzyme 2 Fragments as a Read-Out of Brain Infection in Patients With COVID-19 Encephalopathy

Abstract

Background: This study assesses the cerebrospinal fluid (CSF) levels of the viral receptor angiotensin-converting enzyme 2 (ACE2) and of the serine protease TMPRSS2 fragments in patients with SARS-CoV-2 infection presenting encephalitis (CoV-Enceph).

Methods: The study included biobanked CSF from 18 CoV-Enceph, 4 subjects with COVID-19 without encephalitis (CoV), 21 with non-COVID-19-related encephalitis (Enceph), and 21 neurologically healthy controls. Participants underwent a standardized assessment for encephalitis. A large subset of samples underwent analysis for an extended panel of CSF neuronal, glial, and inflammatory biomarkers. ACE2 and TMPRSS2 species were determined in the CSF by western blotting.

Results: ACE2 was present in CSF as several species, full-length forms and 2 cleaved fragments of 80 and 85 kDa. CoV-Enceph patients displayed increased CSF levels of full-length species, as well as the 80 kDa fragment, but not the alternative 85 kDa fragment, compared with controls and Enceph patients, characterized by increases of both fragments. Furthermore, TMPRSS2 was increased in the CSF of Enceph patients compared with controls, but not in CoV-Enceph patients. The CoV patients without encephalitis displayed unaltered CSF levels of ACE2 and TMPRSS2 species.

Conclusions: Patients with encephalitis displayed an overall increase in CSF ACE2, probably as a consequence of brain inflammation. The increase of the shortest ACE2 fragment only in CoV-Enceph patients may reflect the enhanced cleavage of the receptor triggered by SARS-CoV-2, thus serving to monitor brain penetrance of the virus associated with the rare encephalitis complication. TMPRSS2 changes in the CSF appeared related to inflammation, but not with SARS-CoV-2 infection.

Keywords: ACE2; COVID-19; CSF; TMPRSS2; biomarker.

Graphical Abstract

Figure 1.

Several ACE2 full-length and fragment species coexist in CSF and result altered in patients with encephalitis with and without SARS-CoV-2 infection. A, Schematic representation of ACE2 as a transmembrane type I protein, and of the epitopes recognized by antibodies used in this study (not drawn to scale). ACE2 anti-ectodomain antibodies were AF933 (R&D Systems; goat polyclonal; immunogen, amino acid residues 18–740; 1:200 dilution) and MAB933 (R&D Systems; mouse monoclonal; immunogen, amino acid residues 18–740; 1:500 dilution); anti-C-terminus was antibody ab15348 (Abcam; rabbit polyclonal; immunogen, synthetic peptide corresponding to human ACE2 amino acid residues 788–805; 1:500 dilution). The signal peptide (aa 1–17), carboxypeptidase (aa 18-611), and transmembrane (aa 740–762) domains are represented. The sites of ACE2 shedding with ADAM17 are also indicated (between aa 652–659 and 697–416). The resulting ectodomain fragments are recognized by the AF933 and MAB933 antibodies, but not by the C-terminal antibody ab15348. B, Representative blots of ACE2 immunoreactive species in CSF from nondisease controls using different antibodies, as indicated. Abbreviations: aa, amino acid; ACE2, angiotensin-converting enzyme 2; CSF, cerebrospinal fluid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TM, transmembrane.

Figure 2.

Altered balance of ACE2 fragments is a feature of patients with encephalitis and SARS-CoV-2 infection. A, Levels of CSF ACE2 species were determined in neurologically healthy controls (Ctrl), patients with SARS-CoV-2 infection with encephalitis (CoV-Enceph), patients with encephalitis without SARS-CoV-2 infection (Enceph), and patients with SARS-CoV-2 infection without neurological symptoms (CoV). Samples were blotted with the AF933 antibody against the ectodomain of ACE2. B, Immunoreactivity levels were determined for the 85- and 80-kDa fragments and for the 130-, 105-, and 100-kDa full-length species. C, The relationship between ACE2 fragments, for each sample, are represented by the graph of the quotient obtained by dividing the level of immunoreactivity of the 80 kDa band by the level of immunoreactivity of the 85 kDa band (80/85 kDa). Values are presented as mean ± SEM; P values are indicated; significant changes compared with CoV-Enceph group are highlighted in bold. Abbreviations: ACE2, angiotensin-converting enzyme 2; AU, arbitrary unit; CSF, cerebrospinal fluid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

TMPRSS2 full-length and active fragments coexist in CSF and their relative abundances are altered in patients with encephalitis. A, Schematic representation of TMPRSS2 as a transmembrane type II protein and of the epitopes recognized by the antibodies used in this study (not drawn to scale). TMPRSS2 was resolved with the ectodomain antibody 14437-1-AP (Proteintech; rabbit polyclonal; immunogen, a recombinant protein containing aa 108–492; 1:1000 dilution), the anti-C-terminus antibody H00007113-M05 (Abnova; mouse monoclonal; immunogen, a recombinant protein containing aa 383–492; 1:500 dilution), or the anti-N-terminus antibody OAAB04388 (Aviva Systems Biology; rabbit polyclonal; immunogen, synthetic peptide between aa 1 and 30; 1:1000 dilution). The site of autoproteolytic cleavage at the ectodomain to acquire proteolytic activity is indicated (aa 255–256). The resulting ectodomain fragment is recognized by the 14437-1-AP and C-terminal antibodies, but not by the N-terminal antibody OAAB04388. The transmembrane (aa 84–105) domain is also indicated. B, Representative blots of TMPRSS2 immunoreactive bands present in nondisease CSF samples using different antibodies, as indicated. Abbreviations: aa, amino acid; CSF, cerebrospinal fluid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TM, transmembrane; TMPRSS2, transmembrane protease serine 2.

Figure 4.

Altered balance of TMPRSS2 active fragment in patients with encephalitis. A, CSF samples from neurologically healthy controls (Ctrl), patients with SARS-CoV-2 infection with encephalitis (CoV-Enceph), patients with encephalitis without SARS-CoV-2 infection (Enceph), and patients with SARS-CoV-2 infection without neurological affectation (CoV) were probed with the 14437-1-AP antibody against the ectodomain of TMPRSS2. B, Immunoreactivity levels were determined for the 25-kDa fragment and for the 55-kDa full-length species. C, The relationship between the fragment and full-length species, for each sample, are represented by the graph of the quotient obtained from the 25-kDa compared to the levels of the 55-kDa TMPRSS2 species (25/55 kDa). Values are presented as mean ± SEM; P values are indicated; significant changes compared with CoV-Enceph group are highlighted in bold. Abbreviations: ACE2, angiotensin-converting enzyme 2; AU, arbitrary unit; CSF, cerebrospinal fluid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TMPRSS2, transmembrane protease serine 2.

Figure 5.

Correlation matrices based on Pearson test for all the determined biomarkers from patients with SARS-CoV-2 infection with encephalitis. A, Correlation matrix, corrected for age and sex, of the neurodegeneration markers analyzed in the cohort. B, Correlation matrix, corrected for age, sex, and NfL, of the cytokines and glial markers. Matrices show the value of each of the correlation coefficients; significant correlations are indicated: *P < .5, **P < .05, ***P < .001. C and D, Scatter plot adjusted for age and sex, consistent with the associated correlation matrix, representing correlation of the 80 kDa fragment with (C) sTREM2 and (D) GFAP (solid line represent the correlation, dashed lines represent the 95% confidence interval). Abbreviations: ACE2, angiotensin-converting enzyme 2; Aβ, amyloid-β; AU, arbitrary unit; GFAP, glial fibrillary acidic protein; IL-1β, interleukin 1β; NfL, neurofilament light polypeptide; P-tau, phosphorylated tau; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; sTREM2, soluble triggering receptor expressed on myeloid cells 2; TNF-α, tumor necrosis factor-α; T-tau, total tau.