key: cord-0735098-wx4s1hpm
authors: Bilinska, Katarzyna; von Bartheld, Christopher S.; Butowt, Rafal
title: Expression of the ACE2 virus entry protein in the nervus terminalis reveals the potential for an alternative route to brain infection in COVID-19
date: 2021-06-07
journal: bioRxiv
DOI: 10.1101/2021.04.11.439398
sha: 718d8e9a180fc826720035c8fc8f78ce274138b7
doc_id: 735098
cord_uid: wx4s1hpm

Previous studies suggested that the SARS-CoV-2 virus may gain access to the brain by using a route along the olfactory nerve. However, there is a general consensus that the obligatory virus entry receptor, angiotensin converting enzyme 2 (ACE2), is not expressed in olfactory receptor neurons, and the timing of arrival of the virus in brain targets is inconsistent with a neuronal transfer along olfactory projections. We determined whether nervus terminalis neurons and their peripheral and central projections should be considered as a potential alternative route from the nose to the brain. Nervus terminalis neurons in postnatal mice were double-labeled with antibodies against ACE2 and two nervus terminalis markers, gonadotropin-releasing hormone (GnRH) and choline acetyltransferase (CHAT). We show that a small fraction of CHAT-labeled nervus terminalis neurons, and the large majority of GnRH-labeled nervus terminalis neurons with cell bodies in the region between the olfactory epithelium and the olfactory bulb express ACE2 and cathepsins B and L. Nervus terminalis neurons therefore may provide a direct route for the virus from the nasal epithelium, possibly via innervation of Bowman’s glands, to brain targets, including the telencephalon and diencephalon. This possibility needs to be examined in suitable animal models and in human tissues.

Many previous reports have suggested that the severe acute respiratory syndrome 49 coronavirus 2 (SARS-CoV-2) gains access to the brain by using an olfactory route from , studies suggesting an olfactory route for SARS-CoV-2 to achieve brain 81 infection fail to consider the potential for an alternative route from the nose to the brain, 82 the route via the nervus terminalis. Many peripheral processes of the nervus terminalis 83 innervate the olfactory epithelium, the blood vessels below this epithelium, as well as 84 cells in Bowman's glands (Larsell, 1950) , and the central processes of some of these 85 neurons extend to various targets in the forebrain as far caudal as the hypothalamus 86 (Pearson, 1941; Larsell, 1950; Schwanzel-Fukuda et al., 1987; Demski, 1993 Ridgway et al., 1987) . These properties make 95 the nervus terminalis a strong candidate for expression of ACE2, which is known to 96 regulate blood flow and blood pressure in many tissues (Tikellis and Thomas, 2012) . 97 Expression of ACE2 in the nervus terminalis would suggest that this cranial nerve is a 98 plausible alternative to the olfactory nerve for the SARS-CoV-2 virus to gain access to 99 the brain. However, it has not been previously examined and reported whether nervus Finetek), and cryosectioned at 10-12 μm using a Leica CM1850 cryostat. To verify the specificity of the ACE2 antibody, an ACE2 knock-out (KO) mouse line 121 was obtained from Taconic (strain #18180). Two male homozygous ACE2 KO mice at 122 age 3 weeks old were processed and immunolabeled as described below for wildtype 123 mice. Genotyping was performed according to the manufacturer's suggested PCR 124 protocol. Lack of an ACE2 protein band was confirmed by using Western blots as 125 described previously (Bilinska et al., 2020) . In brief, tissue was homogenized on ice in 126 N-Per Total Protein Extraction reagent (Thermo Scientific) with addition of protease 127 and phosphatase inhibitor cocktails (Sigma-Aldrich). Homogenates were centrifuged 128 for 30 minutes at 20,000 g at 4°C and supernatants were collected. Protein content 129 was measured by the BCA method (Thermo Scientific). Equal amounts of total proteins 130 were mixed with 4x Laemmli sample buffer and boiled for 10 minutes at 80°C. Protein 131 extracts were separated on SDS-PAGE 7.5% gels and mini-protean III apparatus.

GAPDH was used as positive control and to verify equal loading. Proteins were blotted 133 to nitrocellulose membranes using standard Tris-glycine wet method. Membranes 134 were blocked with 5% dry milk (Bio-Rad), incubated with goat polyclonal anti-ACE2 135 (R&D Systems AF3437) at 1/1000 or rabbit polyclonal anti-GAPDH (Protein-Tech, 136 #10494-1-AP) at 1/5000 dilution overnight at 4°C, washed several times in TBST buffer 137 (pH 8.0) and incubated 60 minutes with secondary antibody, anti-goat-HRP (Protein-138 Tech). Signal was detected using Clarity Max chemiluminescence substrate (Bio-Rad).

For confirmation, blots were stripped and re-probed with an additional rabbit 140 monoclonal anti-ACE2 antibody (Abclonal, #A4612). Blots were prepared in three 141 separate experiments with comparable results. Table S2 .

The results were analyzed using GraphPad Prism software. Results are presented as 182 mean ± standard error of the mean (SEM). An unpaired t-test was applied to determine 183 whether the difference in ACE2 colocalization between GnRH + and CHAT + neurons Table S2 . CoV-2 to enter the host cell (Shang et al., 2020) . Our attempts to double-label nervus 270 terminalis neurons using three different anti-TMPRSS2 antibodies (Table S1) suggests that these animals may be more vulnerable to brain infection via the nervus 364 terminaliseven in the absence of an olfactory system. The new coronavirus responsible for the COVID-19 pandemic can infect the brain in 412 humans and in some animal models. It is currently not known how this virus infects the 413 brain. Many researchers believe that the virus enters the brain by using a route along 

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting 601 enzyme 2 transgenic mice

Cloning of the TMPRSS2 gene, which encodes a novel serine protease with 605 transmembrane, LDLRA, and SRCR domains and maps to 21q22

The development of the nervus terminalis in man

Comparison of transgenic and adenovirus hACE2 mouse 613 models for SARS-CoV-2 infection

The 616 terminal nerve in odontocete cetaceans

Localization of choline 619 acetyltransferase and vasoactive intestinal polypeptide-like immunoreactivity in the 620 nervus terminalis of the fetal and neonatal rat

Distribution 623 of luteinizing hormone-releasing hormone in the nervus terminalis and brain of the 624 mouse detected by immunocytochemistry

Cell 627 entry mechanisms of SARS-CoV-2

Neurological infection with SARS-CoV-2 -the story so far

COVID-19 neuropathology at Columbia University Irving Medical 634

New York Presbyterian Hospital. Brain, awab148. Advance online publication

Angiotensin-Converting Enzyme 2 (ACE2) Is a

Key Modulator of the Renin Angiotensin System in Health and Disease

Identification of Transmembrane 642

Protease Serine 2 and Forkhead Box A1 As the Potential Bisphenol A Responsive 643

Furin expression in the nose and olfactory bulb in mice and 647 humans

The olfactory nerve: a shortcut for 650 influenza and other viral diseases into the central nervous system

The terminal nerve and its relation with extrabulbar 654 "olfactory" projections: lessons from lampreys and lungfishes

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung 659 inflammation and impaired function

Acetylcholinesterase and luteinizing hormone-662 releasing hormone distinguish separate populations of terminal nerve neurons

SARS-CoV-2 666 infection causes transient olfactory dysfunction in mice

SARS-CoV-2 670 infects and damages the mature and immature olfactory sensory neurons of hamsters

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice 675 and is a promising target for new drug development

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 680 mice

SARS-CoV-2 spike D614G variant confers enhanced replication and 684 transmissibility. bioRxiv

Neuropathogenesis and Neurologic Manifestations of the Coronaviruses in 689 the Age of Coronavirus Disease 2019: A Review

Schematic sagittal section through a mouse head shows the orientation and 698 planes of tissue sections from Fig. 2A, E, I and M. Sections within those planes were 699 used for demonstration of double-immunolabeling and for cell counting. CNS, central 700 nervous system

in the medial 717 region adjacent to the olfactory bulbs as indicated in Fig. 1. Panels A-D and E-H 718 show slightly different focal planes to demonstrate the morphology of the two or three 719 different neurons

GnRH 722 positive cells in the ACE2 knock-out mouse (I) are not labeled with ACE2 (J)

Control sections probed without primary antibodies or with control rabbit IgG had 725 no detectable signal (not shown). Arrows and triangles indicate double-labeled 726 neurons or lack thereof

Quantification of neurons labeled with nervus terminalis markers, virus 731 entry proteins, and verification of the specificity of the ACE2 antibody

The total number of counted GnRH-positive or CHAT-positive neurons 735 was set at 100%. Error bars represent ± SEM. A t-test shows that the colocalization 736 difference between GnRH-and CHAT-positive nervus terminalis neurons is 737 significant at p<0.0001. For further details, see Table S2. B. Western blot of ACE2 in 738 wildtype (wt) mice and in ACE2 knock-out (KO) mice. The first two lanes (kidney) 739 were loaded with 25 µg total protein, the lanes for olfactory bulb and cerebral cortex 740 were loaded with 60 µg total protein, and probed with the R&D ACE2 antibody. No 741 ACE2 protein was detectable in the ACE2 KO mice, proving that the antibody indeed 742 recognizes ACE2. C-F. Example of GnRH-positive nervus terminalis neurons which 743 are also cathepsin L-positive. C. One GnRH-labeled neuron is marked with a white 744 arrow. D. The same neuron is labeled with the cathepsin L antibody (CatL, white 745 arrow). E. The cell nuclei are stained with Hoechst nuclear dye

Peripheral projections of nervus terminalis (NT) neurons and their 752 presumptive relationship with ACE2-expressing neurons in the olfactory epithelium 753 and known SARS-CoV-2 infection

BG) cells, the olfactory epithelium (OE), and contact cerebrospinal 755 fluid (CSF) spaces. Peripheral projections of NT neurons according to

Cell types 760 that have been documented to be infected by SARS-CoV-2 are indicated with pink 761 asterisks

Ueha et al., 2021) which 765 may facilitate virus entry into those nervus terminalis neurons which innervate BG

GnRH-labeled cells were never 793 labeled for OMP in this region. (E) GnRH-labeled nervus terminalis neurons (one 794 neuron indicated with the white asterisk) did not co-localize with cells positive