Fig 2: Peripheral projections of nervus terminalis (NT) neurons and their presumptive relationship with ACE2-expressing neurons in the olfactory epithelium and known SARS-CoV-2 infection. NT neurons innervate blood vessels (BV), Bowman gland (BG) cells, the olfactory epithelium (OE), and contact cerebrospinal fluid (CSF) spaces. Peripheral projections of NT neurons according to Larsell (1950), CSF contacts according to Jennes (1987). Cells expressing ACE2 are indicated in green, including sustentacular cells (SUS) and BG cells. Both of these cell types have been shown to express ACE2 (Bilinska et al., 2020; Brann et al., 2020; Chen et al., 2020; Ye et al., 2020; Zhang et al., 2020; Klingenstein et al., 2021). Cell types that have been documented to be infected by SARS-CoV-2 are indicated with pink asterisks. SARS-CoV-2 localization in SUS cells according to Bryche et al. (2020); Leist et al. (2020), Ye et al. (2020); Zhang et al. (2020), Zheng et al. (2021); de Melo et al. (2021), and in BG cells according to Bryche et al. (2020); Leist et al. (2020), and Ye et al. (2020). BG cells furthermore express the protease furin (Ueha et al., 2021) which may facilitate virus entry into those nervus terminalis neurons which innervate BG cells.

Fig 3: Examples of double immunofluorescent labeling for nervus terminalis neuronal markers GnRH (A,E) or CHAT (M) and ACE2 (B,F,J,N) in the medial region adjacent to the olfactory bulbs as indicated in Figure 1. (A–H) Show slightly different focal planes to demonstrate the morphology of the two or three different neurons. Nuclei are stained with Hoechst 33258 (C,G,K,O). Merged images are shown in the last column (D,H,L,P). The neuronal somas labeled with GnRH (A,E) Are co-labeled with ACE2 (B,F) as shown after merging (D,H). GnRH positive cells in the ACE2 knock-out mouse (I) are not labeled with ACE2 (J). The majority of cholinergic neurons are not labeled with ACE2 (M,N), as quantified in Figure 3A. Control sections probed without primary antibodies or with control rabbit IgG had no detectable signal (not shown). Arrows and triangles indicate double-labeled neurons or lack thereof. Scale bars: 20 µm.

Fig 4: Examples of double immunofluorescent labeling for nervus terminalis neuronal markers GnRH (A, E) or CHAT (M) and ACE2 (B, F, J, N) in the medial region adjacent to the olfactory bulbs as indicated in Fig. 1. Panels A-D and E-H show slightly different focal planes to demonstrate the morphology of the two or three different neurons. Nuclei are stained with Hoechst 33258 (C, G, K, O). Merged images are shown in the last column (D, H, L, P). The neuronal somas labeled with GnRH (A, E) are co-labeled with ACE2 (B, F) as shown after merging (D, H). GnRH positive cells in the ACE2 knock-out mouse (I) are not labeled with ACE2 (J). The majority of cholinergic neurons are not labeled with ACE2 (M, N), as quantified in Fig. 3A. Control sections probed without primary antibodies or with control rabbit IgG had no detectable signal (not shown). Arrows and triangles indicate double-labeled neurons or lack thereof. Scale bars: 20 µm.

Fig 5: MAP4K3/GLK phosphorylates and stabilizes ACE2 proteins AImmunoblotting analyses of ACE2 and Flag-tagged GLK in HCC827 cells transfected with either Flag-GLK or Flag-GLK (K45E) kinase-dead mutant. For immunoblotting of phosphorylated ACE2, Phos-tagged SDS–PAGE gel (Phos-tag™) was used, followed by immunoblotting with anti-ACE2 antibody. Asterisk denotes unglycosylated ACE2 proteins.B In vitro kinase assays using purified proteins. ACE2 serine phosphorylation, Flag-tagged GLK (anti-GLK), Flag-tagged GLK (K45E) kinase-dead mutant (anti-GLK), and Flag-tagged ACE2 proteins (anti-ACE2) were detected by immunoblotting.CMass spectrometry analysis of GLK-phosphorylated ACE2 proteins after in vitro kinase assays. ACE2 Ser776 and Ser783 residues were phosphorylated by GLK wild-type but not GLK (K45E) kinase-dead mutant.DMass spectrometry analysis of the ACE2 peptides from the serum exosomes of COVID-19 patients. The ACE2 protein peptide sequences containing phospho-Ser776 or phospho-Ser783 residue of ACE2 proteins detected in the serum exosomes of COVID-19 patients (Cohort #3) are shown. Exosomes were isolated using ExoQuick kits, and soluble proteins were removed by ExoQuick ULTRA columns. Exosomes were further purified by immunoprecipitation using a combination of anti-CD9, anti-CD63, and CD81 magnetic beads.E In vitro kinase assays using immunoprecipitated Flag-tagged ACE2 or Flag-tagged GLK immunocomplexes. ACE2 serine phosphorylation, ACE2, and GLK were detected by immunoblotting using anti-phospho-serine, anti-ACE2, and anti-GLK antibodies, respectively.FImmunoblotting analyses of Myc-tagged GLK, Flag-tagged ACE2, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual ACE2 (S776/783A, S776A, or S783A) mutants plus either empty vector or Myc-GLK.GImmunoblotting analyses of Flag-tagged ACE2 and tubulin proteins in HEK293T cells transfected with Flag-ACE2 wild-type or individual phosphomimetic ACE2 (S776/783E, S776E, or S783E) mutants.HFlag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells transfected with Flag-ACE2 wild-type or a phosphomimetic ACE2 (S776/783E) mutant, followed by immunoblotting with anti-Lys48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 µM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three (A, B, E, F, G, H) independent experiments. Source data are available online for this figure.