SARS-CoV-2 spike protein induces cognitive deficit and anxiety-like behavior in mouse via non-cell autonomous hippocampal neuronal death

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is accompanied by chronic neurological sequelae such as cognitive decline and mood disorder, but the underlying mechanisms have not yet been elucidated. We explored the possibility that the brain-infiltrating SARS-CoV-2 spike protein contributes to the development of neurological symptoms observed in COVID-19 patients in this study. Our behavioral study showed that administration of SARS-CoV-2 spike protein S1 subunit (S1 protein) to mouse hippocampus induced cognitive deficit and anxiety-like behavior in vivo. These neurological symptoms were accompanied by neuronal cell death in the dorsal and ventral hippocampus as well as glial cell activation. Interestingly, the S1 protein did not directly induce hippocampal cell death in vitro. Rather, it exerted neurotoxicity via glial cell activation, partially through interleukin-1β induction. In conclusion, our data suggest a novel pathogenic mechanism for the COVID-19-associated neurological symptoms that involves glia activation and non-cell autonomous hippocampal neuronal death by the brain-infiltrating S1 protein.


SARS-CoV-2 spike protein induces cognitive decline and anxiety-like behavior in mice.
To test whether the brain-infiltrating SARS-CoV-2 S1 protein is involved in the neurological problems observed in COVID-19 patients, we directly introduced S1 proteins into the dorsal hippocampus, a brain sub-region critical for cognition and emotion 10 , of mice and subjected the mice to a series of behavioral tests to measure cognitive and affective brain functions (Fig. 1a). In novel object recognition and novel location tests, the S1 protein-injected mice exhibited reduced discrimination capacity compared to the vehicle-injected control mice (Fig. 1b). In contrast, locomotive function, which was measured by the total distance traveled during the behavioral session, was not significantly altered. These results indicate that the S1 protein of SARS-CoV-2 in the hippocampus affected mouse cognitive brain function. In the elevated plus maze test, the S1 proteininjected mice spent less time in the center and explored more in the closed arm compared to the control group (Fig. 1c). In addition, in the open field test, the S1 protein-injected group spent less time in the center of the chamber and spent more in the periphery compared to control mice, manifesting anxiety-like behavior (Fig. 1d). Taken together, the hippocampal injection of SARS-CoV-2 S1 protein leads to cognitive deficits and anxiety-like behavior in mice.

SARS-CoV-2 spike protein induces hippocampal neuronal death and glia activation.
We then evaluated whether the SARS-CoV-2 spike protein can lead hippocampal neuronal cell death. In cresyl violet staining, a markedly reduced neuronal cell density was observed in the CA1 and DG areas of the dorsal and ventral hippocampus in the S1 protein-injected mice (Fig. 2a). The effects of SARS-CoV-2 S1 protein on hippocampal neuronal death was further confirmed by immunohistochemistry (Fig. 2b). Quantifying NeuN-positive neurons demonstrated that S1 protein injection reduced dorsal hippocampal neurons by 35% in both the CA1 and the DG regions and reduced ventral hippocampal neurons by 20% (Fig. 2c). These results show that the SARS-CoV-2 S1 protein induced hippocampal neuronal cell death and implies that the brain-penetrating SARS-CoV-2 spike protein may exert neurotoxic effects in COVID-19 patients.
A recent clinical study described an increased glial cell number as well as decreased neuronal cell density in the brains of COVID-19 patients 11 . Therefore, we assessed the effects of SARS-CoV-2 S1 protein on glial cell activation by immunohistochemistry using GFAP and Iba-1 antibodies, which are specific markers for astrocytes and microglia, respectively. Following S1 protein administration in the dorsal hippocampus, GFAP-immunoreactive astrocytes were notably increased in the dorsal hippocampus (Fig. 3a). The fluorescence intensity of GFAP signals increased by 59 and 63% in both the dorsal and ventral hippocampus of CA1 and DG regions (Fig. 3b). Similarly, a dramatic increase in Iba-1 immunoreactivity was detected in the dorsal hippocampus of S1 protein-injected mice (Fig. 3c). Morphologically, the microglia in the S1 protein-injected mice manifested more circular cell bodies and shorter branch lengths than the microglia in the control mice; these are key morphological features of reactive microglia 12 (Fig. 3d). Taken together, our data demonstrate that the administration of SARS-CoV-2 S1 protein to the hippocampus induces glial cell activation as well as neuronal cell death.
IL-1β released from the activated glia induce hippocampal neuronal cell death. Next, the neurotoxic mechanism of the SARS-CoV-2 spike protein was investigated by treating hippocampal neurons with the S1 protein in vitro. Incubation with S1 protein (5 µg/ml) for 12 h did not affect the survival rate of primary cultured hippocampal neurons (Fig. 4a), suggesting the non-cell autonomous neurotoxicity of the SARS-CoV-2 spike protein. We then incubated primary hippocampal neurons in the conditioned medium from mixed glia that were stimulated with S1 protein to test whether S1 protein-induced neuronal cell death was mediated by activated glia. After 12 h of incubation in the conditioned medium, the hippocampal neuronal cell number was significantly decreased (Fig. 4b), indicating that certain factor(s) derived from the S1 protein-activated glial cells induced neuronal cell death. Studies have shown that glial cells produce neurotoxic molecules such as pro-inflammatory cytokines under pathogenic conditions 13 . The pro-inflammatory cytokine expression in the primary glia upon SARS-CoV-2 S1 protein stimulation was tested to identify the putative molecules that are derived from the activated glia and mediate hippocampal neuronal death. The transcription of IL-1β, a potentially neurotoxic inflammatory cytokine 14 , was induced in primary glial cells by S1 protein stimulation (Fig. 4c). In addition, the release of IL-1β into conditioned media from the activated primary glial cells was also confirmed (Fig. 4d). IL-1β expression was also up-regulated more than sevenfold in dorsal as well as ventral hippocampi by S1 protein injection in vivo (Fig. 4e). These data suggest that the IL-1β released by spike protein-activated glia may induce hippocampal neuronal cell death. This was tested by incubating the conditioned media with IL-1β-neutralizing antibody before transfer to the primary hippocampal neurons. Depleting IL-1β from the conditioned media almost completely rescued the S1 protein-mediated hippocampal neuronal cell death (Fig. 4f). Taken together, our data indicate that the SARS-CoV-2 spike protein causes non-cell autonomous hippocampal neuronal cell death by inducing IL-1β expression from glial cells.

Discussion
Many clinical reports of COVID-19 survivors suffering from various neurological and psychiatric symptoms such as cognitive decline and mood disorder undoubtedly show that SARS-CoV-2 affects the human CNS. The initial assumption of direct SARS-CoV-2 infection in the brain was not supported by following studies; thus, the cellular and molecular mechanisms are still elusive. The recent discovery of the blood-brain barrier-penetrating SARS-CoV-2 spike protein 9 offers a new possibility that the SARS-CoV-2-derived spike protein causes neurological or psychiatric symptoms observed in the COVID-19 survivors, which we tested in this study. We demonstrated that S1 protein administration into mice hippocampi led to cognitive decline and anxiety-like behavior. These data www.nature.com/scientificreports/ Figure 1. Administration of SARS-CoV-2 S1 protein induces cognitive deficit and anxiety-like behavior in mice. (a) Experimental design of S1 administration and behavioral tests. S1 protein (n = 10) or saline (Control, n = 10) was administered to C57BL/6 mice at 8 weeks of age, and behavioral tests were started 1 week after the administration. (b) Cognitive deficits were assessed using novel object recognition (NOR, left) or novel location recognition (NLR, right) tests and are presented as discrimination index percentages. Locomotor activity was measured by the total distance moved by mice in the chamber within a test session (bottom). The exploration time of a novel object or location divided by total exploration time was presented as the discrimination index of the novel object or location. The pink circle indicates a novel object or novel location. www.nature.com/scientificreports/ www.nature.com/scientificreports/  www.nature.com/scientificreports/ suggest that several neurological and psychiatric symptoms observed in COVID-19 patients can be mediated by the brain-penetrating S1 protein in the absence of direct CNS infection of SARS-CoV-2.
It is well known that the hippocampus is important for cognition and emotions 15,16 . Specifically, the dorsal hippocampus is involved in cognition as well as learning and memory 17,18 and the ventral hippocampus in affective brain functions such as anxiety 19 . Therefore, it is easily conceivable that hippocampal neuronal cell death in these areas due to S1 protein administration may lead to the cognitive deficits and anxiety-like behavior observed in our study. Notably, angiotensin-converting enzyme 2 (ACE2) and neuropilin-1, two receptors for S1 protein, are relatively highly expressed in the hippocampus as compared to other brain regions 20,21 . This may increase the susceptibility of the hippocampus to the S1 protein effects and might explain why hippocampus-dependent neurological and psychological symptoms such as cognitive deficit or anxiety and depression often manifest in COVID-19 patients.
In our effort to elucidate the mechanisms, we found that S1 protein induced non-cell autonomous hippocampal neuronal cell death. The S1 protein exerted hippocampal neurotoxicity via activating glial cells. More specifically, we found that IL-1β expressed by the S1 protein-activated glia contributes to hippocampal neuronal cell death. The inflammatory activation of glia and subsequent bystander neuronal cell death have been often observed in other neurotropic virus infections such as HIV and Japanese encephalitis virus infection, in which glia-expressed IL-1β was implicated [22][23][24] . Previously, the S1 protein of coronavirus was shown to induce inflammatory cytokines by activating NF-κ B signaling in microglia 25 , and ACE2 and CD147, receptors that interact with S1, are expressed on glial cells 20,26 . Taking into account these prior studies, our data suggests that the brainpenetrating S1 protein of SARS-CoV-2 can activate hippocampal glial cells to express IL-1β and thereby induce bystander hippocampal neuronal cell death.
In summary, we present evidence that the S1 protein of SARS-CoV-2 introduced into the hippocampus can induce non-cell autonomous hippocampal neuronal death resulting in cognitive deficit and anxiety-like behavior. This novel mechanism may open a new avenue for the treatment of neurological and psychological symptoms of COVID-19 survivors in the future.

Materials and methods
Animals. Eight-to-ten weeks old male C57BL/6 mice, weighing 21-25 g, were obtained from Daehan Biolink Co. Ltd. (Chungbuk, Korea) and randomly divided into animal cages. Animals were housed and maintained in a controlled environment at 22-24 °C and 55% humidity with 12 h light/dark cycles and fed regular rodent chow and tap water ad libitum. All animal care was guided by the Seoul National University Institutional Animal Care and Use Committee (SNU IACUC) to minimize pain and distress during experimental interventions and all animal experimental procedures were performed following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
Stereotaxic spike protein delivery to mice hippocampi. Following a previous study, hippocampal administration of S1 protein was performed 27 . Eight-week-old mice were used for the spike protein injections. S1 protein was purchased from Acrobiosystems (Cat #S1N-C52H4, Newark, DE, USA), and used after polymixin B (Cat #P1004, Sigma-Aldrich, St. Louis, MO, USA) treatment (30 µg/ml). The animals were anesthetized with isoflurane, and the injection paths were drilled into their skulls. Then, 5 µg of S1 protein (1 µg/µl) were bilaterally injected into each hippocampal region using a Hamilton syringe (Cat #80330, Hamilton Company, Reno, NV, USA) attached to a syringe pump (Cat #53311, Stoelting Co., Wood Dale, IL, USA) at a constant volume of 0.5 µl/min. The injection coordinates of the hippocampus were 1.5 (ML), − 2.06 (AP), and − 2.0 (DV) from the bregma.
Novel object recognition and location recognition test. The novel object recognition and location recognition tests were performed using previously described methods 28,29 with minor modifications. First, each mouse was placed on one side of the open field box and allowed to freely explore for 10 min. After one day, two identical objects were presented to the mouse for 10 min. Then, one object was replaced with a novel object, and the mouse was allowed to explore for 5 min. Subsequently, one object was replaced in a novel location, and the mouse was allowed to explore for 5 min. The chamber and objects were cleaned with 70% ethanol between trials to remove olfactory stimuli. Testing sessions were video monitored, and object exploration times were scored by a blinded experimenter. Results are expressed as the discrimination ratio of time spent with the novel object or novel location to the total exploration time. The discrimination index of the novel object or location was calculated by the following formula: (discrimination time of novel object or location/total discrimination time of novel and familiar object or location)*100. The exploration time were measured automatically by SMART 3.0 software (Panlab, Barcelona, Spain).

Elevated plus maze (EPM).
The EPM was used to examine anxiety-like behavior. The behavioral apparatus consisted of two open arms and two closed arms elevated 50 cm above the floor. Mice were placed individually in the center of the maze, facing an open arm, and allowed to freely explore for 5 min. The maze was cleaned with 70% ethanol after each test to prevent influence from the previously tested mouse. The times spent in the open and closed arms were measured automatically by SMART 3.0 software.
Open field test (OFT). The OFT was performed as previously described 30 . Each mouse was placed in the center of an open arena and allowed to freely explore the arena for 5 min. The times spent in the center and the periphery zone were automatically measured by SMART 3.0 software. www.nature.com/scientificreports/ Cresyl violet staining. Cresyl violet (Cat #C5042, Sigma-Aldrich) staining was performed as previously described with minor modification 31 . Briefly, brain sections were rehydrated with serially diluted ethanol (from 100 to 70%) for 2 min and with xylene for 5 min. Rehydrated tissues were incubated with 0.1% cresyl violet solution for 10 min. After the solution was washed away with distilled water, tissues were dehydrated with diluted ethanol (from 70 to 100%) for up to 1 min for each. The samples were rinsed in xylene and mounted on slides.
Immunohistochemistry. Immunostaining was carried out using previously established protocols 32 .

Microglia morphological analysis.
Morphological analysis of microglia was performed using a method of MATLAB (version R2021a, The MathWorks Inc., Natick, MA, USA) as described previously 33 . Briefly, by tracking microglial soma and processes, intensity quantiles across the image were identified, and we then quantified the resultant image. To normalize fluorescence intensities between tissue samples, the quantile level of the background and the soma intensity were automatically modified. Minimum object recognition (i.e., soma) was set to 200-300 pixels. At 20 × magnification, the total area of view (320 µm 2 ) of the region of interest (hippocampal CA1 and DG) was evaluated.